AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 e...AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.展开更多
AIM:To investigate the influence of ophthalmic viscoelastic devices(OVDs)and different surgical approaches on the intraocular pressure(IOP)before and after creation of the curvilinear circular capsulorhexis(CCC)as a m...AIM:To investigate the influence of ophthalmic viscoelastic devices(OVDs)and different surgical approaches on the intraocular pressure(IOP)before and after creation of the curvilinear circular capsulorhexis(CCC)as a measure for anterior chamber stability during this maneuver.METHODS:Prospective experimental WetLab study carried out on enucleated porcine eyes.IOP was measured before and after CCC with the iCare Rebound tonometer(iCare ic200;iCare Finland Oy,Vantaa,Finland).The OVDs used were a cohesive one[Z-Hyalin,Carl Zeiss Meditec AG,Germany;hyaluronic acid(HA)]and a dispersive[Z-Celcoat,Carl Zeiss Meditec AG,Germany;hydroxy propylmethylcellulosis(HPMC)].The CCC was created using Utrata forceps or 23 g microforceps in different combinations with the OVDs.RESULTS:Using the Utrata forceps the IOP dropped from 63.65±6.44 to 11.25±3.63 mm Hg during the CCC.The use of different OVDs made no difference.Using the 23 g microforceps the IOP dropped from 65.35±8.15 to 36.55±6.09 mm Hg.The difference between IOP drop using either Utrata forceps or 23 g microforceps was highly significant regardless of the OVD used.CONCLUSION:Using the sideport for the creation of the capsulorhexis leads to a lesser drop in IOP during this maneuver compared to the main incision in enucleated porcine eyes.The use of different OVD has no significant influence on IOP drop.展开更多
Sertoli cells are indispensable for guaranteeing normal spermatogenesis and male fertility.Although a huge number of long non-coding RNAs(lncRNAs)are identified from developing porcine testicular tissues and have been...Sertoli cells are indispensable for guaranteeing normal spermatogenesis and male fertility.Although a huge number of long non-coding RNAs(lncRNAs)are identified from developing porcine testicular tissues and have been predicted with crucial regulatory roles in spermatogenesis,their functions and regulatory mechanisms are still in infancy.Herein,we mainly explored the regulatory and functional roles of lncFPFSC in proliferation and apoptosis of immature porcine Sertoli cells.The results demonstrated that lncFPFSC was predominantly located in the cytoplasm of immature porcine Sertoli cells.lncFPFSC overexpression promoted cell cycle progression and cell proliferation,as well as inhibited cell apoptosis,whereas siRNA-induced lncFPFSC knockdown resulted in the opposite effects.Mechanistically,lncFPFSC acted as a sponge for miR-326.Overexpression of miR-326 inhibited cell proliferation and induced cell apoptosis,which further abolished the effects of lncFPFSC overexpression.The euchromatic histone-lysine N-methyltransferase 2(EHMT2)gene was directly targeted by miR-326,and its mRNA and protein expressions were both negatively regulated by miR-326 in immature porcine Sertoli cells.Then,siRNA-induced EHMT2 knockdown resulted a similar effect of miR-326 inhibition.Collectively,lncFPFSC promoted proliferation and inhibited apoptosis in immature porcine Sertoli cells through modulating the miR-326/EHMT2 axis.This study expanded our understanding of non-coding RNAs in participating porcine spermatogenesis through deciding the fate of Sertoli cells,and the competing endogenous RNA(ceRNA)network,and provided new molecular markers to treat Sertoli cell disorder inducing male infertility.展开更多
Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Lut...Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.展开更多
[Objective]The paper was to identify,isolate,and characterize porcine astrovirus in Shandong Province between 2021 and 2023.[Method]A total of 1025 samples of porcine diarrhea samples were collected from various regio...[Objective]The paper was to identify,isolate,and characterize porcine astrovirus in Shandong Province between 2021 and 2023.[Method]A total of 1025 samples of porcine diarrhea samples were collected from various regions of Shandong Province between January 2021 and October 2023.The samples were tested by RT-PCR,followed by sequencing and phylogenetic analyses of the polymerase.[Result]The total positive rate of PAstV was 34.6%(355/1025).The respective proportions of individuals infected with PAstV-1,PAstV-2,PAstV-4 and PAstV-5 were 25.4%(90/355),28.2%(100/355),35.2%(125/355)and 22.5%(80/355),respectively.Additionally,mixed infection was observed.Meanwhile,849 samples of healthy pigs were tested by RT-PCR,and the results demonstrated that the total positive rate of PAstV was 8.13%(69/849).Of these,the proportion of PAstV-1,PAstV-2 and PAstV-4 infection was 27.5%(19/69),37.7%(26/69)and 40.6%(28/69),and a mixed infection also existed.Further sequencing and characterization of some the selected isolates revealed low sequence identities(56.2%)with known PAstV strains,indicating the presence of novel types or genotypes of PAstVs.Furthermore,the isolation conditions of porcine astrovirus were optimized,resulting in the purification of a pure PAstV-4 strain(designated PAstV-4-GRF1).The virus was found to exhibit typical astroviral morphology,with nucleotide identity ranging from 89.9 to 95.4%with previously published PAstV-4 strains.Then,macrovirus transcriptome sequencing showed that 88.30%of the CRF1 samples were mammalian astroviruses.By species classification,PAstV 4 and PAstV 2 accounted for 21.79%and 0.32%,respectively.Phylogenetic tree analysis showed that the c15050 fragment was identical to the GRF-1 sequencing fragment of the isolated strain,and exhibited the highest homology with the Hunan PAstV-4 sequence MK460231 in China.[Conclusion]As the inaugural isolated PAstV-4 strain,it furnishes pivotal materialfor the investigation ofthe biological and pathogenic properties of this virus as well as for the prospectivedevelopment of relevant biological and diagnostic reagents.展开更多
Objective Endoscopic tympanoplasty includes various surgical methods,such as internal repair,interlayer repair,and external overlay.This technique requires autologous materials,allografts,and xenografts,which are used...Objective Endoscopic tympanoplasty includes various surgical methods,such as internal repair,interlayer repair,and external overlay.This technique requires autologous materials,allografts,and xenografts,which are used to repair tympanic membrane(TM)perforation.To obtain good results,appropriate surgical methods and repair materials should be selected.This study aims to assess the efficacy of repairing refractory TM perforations in the porcine small intestinal submucosa(SIS)during transcanal endoscopic type I tympanoplasty.Method A retrospective chart review was performed on patients who underwent TM perforation repair with porcine SIS and tragus cartilage between January 2022 and September 2022 at Sir Run Run Shaw Hospital,Zhejiang University School of Medicine.Perforation size,tympanic status,pre-and postoperative symptoms,follow-up data,wound healing rates,and hearing improvement were analysed.Results Of the 115 patients included in the study,56 underwent interlayer repair with porcine SIS of the TM,and 59 patients underwent internal repair with tragus cartilage.No significant difference was found between the two groups at baseline in terms of age,sex,disease course,perforation side,tympanic status,underlying disease,or preoperative infection.The total postoperative effective rate of interlayer implantation with porcine SIS was 91.07%(51 patients),and that of internal implantation with tragus cartilage was 88.14%(52 patients).No significant difference was found in terms of the graft success rate between the two surgical methods(p=0.887).Postoperative pure tone auditory(PTA)and air-bone gap(ABG)density significantly increased in both groups compared with before surgery(p<0.05).However,the postoperative PTA and ABG density were not significantly different 3 months post-surgery between the two groups(p>0.05).Compared to those in the internal implantation group,the patients in the interlayer group had a shorter operation duration(51.36±6.76 min vs.59.71±7.45 min,t=6.298,p<0.001)and less blood loss(11.91±2.61 mL vs.15.27±2.57 mL,t=7.019,p<0.001).Conclusions Our study suggests that the porcine SIS,as well as the tragus cartilage,has a high success rate in repairing irreversible TM perforation.Endoscopic tympanoplasty via interlayer implantation with porcine SIS offers distinct advantages,including the absence of donor-site incision and scar formation,and ease of graft modification and manipulation.展开更多
[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS). [Method] Antigen location and hist...[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS). [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.展开更多
To observe the acute toxicity of recombinant porcine interferen-alpha (IFN-alpha) in mice and thus provide a basis for the clinical safety. [Method] According to the principles of acute toxicity, all the mice were d...To observe the acute toxicity of recombinant porcine interferen-alpha (IFN-alpha) in mice and thus provide a basis for the clinical safety. [Method] According to the principles of acute toxicity, all the mice were divided into two major groups (intraperitoneally injected group and intramuscularly injected group) respectively at high dose, moderate dose and low dose. And the normal control group was also set up. Within 14 d after administration, the behavior of mouse and the degree of toxicity were continuously observed. The hematological indexes and biochemical indexes of blood were detected to obtain the preliminary toxicity data of the recombinant porcine IFN-alpha. And at the end of the experiment, mice were sacrificed for autopsy. [ Result] There was not significant difference in external performance, behavioral characteristics, body temperature, weight, pathological anatomy of visceral organs, hematological indexes and biochemical indexes between the experimental groups and the control group. [ Conclusion] The highest dose of porcine interferon (5.0 x 10s IU per mouse) in this experiment or the dose lower than this dosage should not have significant toxic effects on mice, and the recombinant porcine IFN-alpha is safe in clinical application.展开更多
Three pairs of primer were designed for amplification of porcine pseudorabies virus (PRV) gB, gE, and TK gene by multiplex PCR (multi-PCR) in order to differentiate vaccine strains from field isolates. Three speci...Three pairs of primer were designed for amplification of porcine pseudorabies virus (PRV) gB, gE, and TK gene by multiplex PCR (multi-PCR) in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp (gB gene), 298 bp (gEgene), and 208 bp (TKgene), Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.展开更多
[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analYze the immunological activi...[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 (DE3) and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 (DE3). Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.展开更多
[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological...[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological method. [Method] The recombinant expression plasmid pGEX-6P-5 was transformed into BL21 and expressed after being induced with IPTG. The solubility analysis of expression products was carried out, and then the recombinant protein was purified for SDS-PAGE identification and Western-blot analysis. Finally, the recombinant antigen was used in the immune experiment of guinea pigs. [Result] The target protein content accounted for 30% of the total cells protein content according to the chromatography scanning, and the purity of target protein after purification reached 80%. The purified protein was analyzed by Western-blot and immune experiment of guinea pigs, and the results showed that the expressed protein had good reactionogenicity and immunogenicity. [Conclusion] This study provides materials for further studies on the function between PRRSV ORF5 gene and its editing protein, which also lays a foundation for porcine reproductive and respiratory syndrome virus genetic engineering products.展开更多
[ Objective] The aim of this study was to provide a basis for study on adiponectin as a candidate gene for fat deposition. [ Method] The promoter sequence of adiponectin was obtained by porcine BAC library screening a...[ Objective] The aim of this study was to provide a basis for study on adiponectin as a candidate gene for fat deposition. [ Method] The promoter sequence of adiponectin was obtained by porcine BAC library screening and primer-walking method. The polymorphisms of adiponectin promoter from 290 pigs, including 5 breeds of Lantang pig, Large spotted pig, Large white pig, Landrace and Duroc, were analyzed with PCR-RFLP. [ Result] At SNP site of adiponectin 5'-flanking region -1 010 bp (G/A), GG genotype frequency in Chinese indigenous pigs was significantly higher than that in exotic pigs. At SNP site of adiponectin 5'-flanking region -394 bp (T/C), the genotype distribution of Chi- nese indigenous pigs was abundant, while no CC genotype was detected in exotic pigs, and T allele frequency was higher in exotic pigs. [ Conclusion] SNP site mutation of - 1 010 bp (G/A) may lead to changes of the gene transcription level, while SNP site of -394 bp (T/C) properly has no relationship with gene transcription level and fat deposition.展开更多
[ Objective] The aim was to analyze the reason and epidemic trend of PRRSV, and provide theoretical basis for preventing and controlling PRRS. [Methed]According to the sequence of ATCC VR-2332 strain porcine reproduct...[ Objective] The aim was to analyze the reason and epidemic trend of PRRSV, and provide theoretical basis for preventing and controlling PRRS. [Methed]According to the sequence of ATCC VR-2332 strain porcine reproductive and respiratory syndrome virus published by the GenBank, the primers were designed and synthesized. ORF5 gene sequences of seven prevalence strains were amplified by RT-PCR. The sequences of ORF5 genes were analyzed by DNAStar and compared with those of ATCC VR-2332, CH-1 a, B J-4, LV-M96262 and MLV vaccine strains, phylogenetic tree among isolates was analyzed. [Result] Analysis of nucleotide sequence showed that the homology was 88.1% - 98.8%, 89.9% -95.2%, 85.6% -98.7% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, BJ-4, and the homology was 54.7% -56.9% between ORF5 genes and LV. Analysis of amino acid sequence showed that the homology was 88.1% -96.8%, 88.1% - 94.5%, 86.1% -96.5% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, bBJ-4, the homology was 54.7% -56.2% between the ORF5 genes and LV.[ Conclusion] The variation of prevalence strains was great in the ORF5 gene region, the homology of ORF5 gene sequence was higher and genetic relationship was nearer during prevalence strains in the same region, or was far in different regions.展开更多
[ Objective] To clone the porcine interleukin-18(/L-18) cDNA and explore the immunological effectiveness of porcine IL-18 as an adjuvant of genetic vaccine. [ Method] The spleen lymphccytes were isolated from Henan ...[ Objective] To clone the porcine interleukin-18(/L-18) cDNA and explore the immunological effectiveness of porcine IL-18 as an adjuvant of genetic vaccine. [ Method] The spleen lymphccytes were isolated from Henan three-way cross-breeding pigs. According to the porcine IL-18 gene in GenBank, a pair of specific primers was designed. The full length cDNA of porcine IL-18 was amplified by RT-PCR. Subsequently, porcine IL-18 cDNA was cloned into pGEM-T vector and sequenced and analyzed. [ Result] The porcine IL-18 gene demonstrated an open reading frame of 579 bp encoding an inactive precursor protein with 192 amino acids. The precursor protein had no typical hydrophobic signal peptide and cleaved by interleukin-1 beta (IL-1β) converting enzyme(ICE) in caspase-1 splice site; the porcine mature protein had biological activity: After comparing with other porcine IL-18 genes, the nucleotide sequence homology was over 96% and the deduced amino acid homology was more than 98%. [ Conclusion] A full length procine IL-18 gene was gained. It lays the foundation for porcine IL-18 as an adjuvant of genetic vaccine.展开更多
To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were...To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were then identified by immunohistochemical staining, and their culture viability measured through the MTT method in vitro. This induced them to differentiate into endocrine cells and detect their function. The isolated IPSCS did not express nestin, but expressed CK-19, a marker of ductal epithelia cells and ct-actin, a smooth muscle marker, demonstrating the growth characteristics of ES-like cells, and strong proliferative ability, after 18 passages. They could excrete insulin, and showed ultrastructure changes after being induced. Porcine pancreatic stem cells can be isolated by this method, induced to form islet-like clusters, and can secret insulin.展开更多
AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population o...AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.展开更多
AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix(APCM) in vitro.·METHODS:The scaffold w...AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix(APCM) in vitro.·METHODS:The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate(SDS)solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin(HE)staining and 4’,6-diamidino-2-phenylindole(DAPI)staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS:Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION:Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.展开更多
[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus(PPV). [ Method] One pair of primers were designed according to PPV genome sequences on...[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus(PPV). [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a ( + ) with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.展开更多
[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porci...[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porcine fetal fibroblast cells,which were normally grown and passed,were collected before total RNA was extracted respectively.The expression ofAPOEgene in different generations of porcine fetal fibro-blast cells was detected by RT-PCR technique.[Result]The expression level of porcine APOE mRNA in the first generation of porcine fetal fi-broblast cells was the highest,and then it gradually decreased with the increase of cell generations and was the lowest in the fiftieth generation.[Conclusion]The expression ofAPOEgene had the selective trend in different generations of porcine fetal fibroblast cells.展开更多
Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic ...Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China's pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses (3×103-3×105 TCID50).The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever (41.0-41.9oC) and high morbidity and mortality (60-100%),the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection (DPI).The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-g and IL-10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.展开更多
基金Key R&D Plan of Shaanxi Province(No.2021SF-331).
文摘AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.
文摘AIM:To investigate the influence of ophthalmic viscoelastic devices(OVDs)and different surgical approaches on the intraocular pressure(IOP)before and after creation of the curvilinear circular capsulorhexis(CCC)as a measure for anterior chamber stability during this maneuver.METHODS:Prospective experimental WetLab study carried out on enucleated porcine eyes.IOP was measured before and after CCC with the iCare Rebound tonometer(iCare ic200;iCare Finland Oy,Vantaa,Finland).The OVDs used were a cohesive one[Z-Hyalin,Carl Zeiss Meditec AG,Germany;hyaluronic acid(HA)]and a dispersive[Z-Celcoat,Carl Zeiss Meditec AG,Germany;hydroxy propylmethylcellulosis(HPMC)].The CCC was created using Utrata forceps or 23 g microforceps in different combinations with the OVDs.RESULTS:Using the Utrata forceps the IOP dropped from 63.65±6.44 to 11.25±3.63 mm Hg during the CCC.The use of different OVDs made no difference.Using the 23 g microforceps the IOP dropped from 65.35±8.15 to 36.55±6.09 mm Hg.The difference between IOP drop using either Utrata forceps or 23 g microforceps was highly significant regardless of the OVD used.CONCLUSION:Using the sideport for the creation of the capsulorhexis leads to a lesser drop in IOP during this maneuver compared to the main incision in enucleated porcine eyes.The use of different OVD has no significant influence on IOP drop.
基金supported by the special funds for Changsha Municipal Natural Science Foundation,China(kq2202229)the Hunan Provincial Natural Science Foundation of China(2023JJ30296 and 2023JJ60247)a Key R&D Projects(2020NK2024)in Hunan Province,China(2020NK2024)。
文摘Sertoli cells are indispensable for guaranteeing normal spermatogenesis and male fertility.Although a huge number of long non-coding RNAs(lncRNAs)are identified from developing porcine testicular tissues and have been predicted with crucial regulatory roles in spermatogenesis,their functions and regulatory mechanisms are still in infancy.Herein,we mainly explored the regulatory and functional roles of lncFPFSC in proliferation and apoptosis of immature porcine Sertoli cells.The results demonstrated that lncFPFSC was predominantly located in the cytoplasm of immature porcine Sertoli cells.lncFPFSC overexpression promoted cell cycle progression and cell proliferation,as well as inhibited cell apoptosis,whereas siRNA-induced lncFPFSC knockdown resulted in the opposite effects.Mechanistically,lncFPFSC acted as a sponge for miR-326.Overexpression of miR-326 inhibited cell proliferation and induced cell apoptosis,which further abolished the effects of lncFPFSC overexpression.The euchromatic histone-lysine N-methyltransferase 2(EHMT2)gene was directly targeted by miR-326,and its mRNA and protein expressions were both negatively regulated by miR-326 in immature porcine Sertoli cells.Then,siRNA-induced EHMT2 knockdown resulted a similar effect of miR-326 inhibition.Collectively,lncFPFSC promoted proliferation and inhibited apoptosis in immature porcine Sertoli cells through modulating the miR-326/EHMT2 axis.This study expanded our understanding of non-coding RNAs in participating porcine spermatogenesis through deciding the fate of Sertoli cells,and the competing endogenous RNA(ceRNA)network,and provided new molecular markers to treat Sertoli cell disorder inducing male infertility.
基金supported by the Korea Research Institute of Bioscience and Biotechnology(KRIBB)Research Initiative Program(KGM4252331,KGM5382322),Republic of Korea.
文摘Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.
基金Supported by Sub-project of the National Key Research and Development Program(2023YFD1800804-06)National Natural Science Funds of China(32302918 and 32302919)+2 种基金Innovation Capacity Improvement Project for Technology-based SMEs in Shandong Province(2023TSGC0006)Science and Technology Cooperation Project of Shandong and Chongqing(2022LYXZ030)Shandong Province Key R&D Program Rural Revitalization Project(2023TZXD083)。
文摘[Objective]The paper was to identify,isolate,and characterize porcine astrovirus in Shandong Province between 2021 and 2023.[Method]A total of 1025 samples of porcine diarrhea samples were collected from various regions of Shandong Province between January 2021 and October 2023.The samples were tested by RT-PCR,followed by sequencing and phylogenetic analyses of the polymerase.[Result]The total positive rate of PAstV was 34.6%(355/1025).The respective proportions of individuals infected with PAstV-1,PAstV-2,PAstV-4 and PAstV-5 were 25.4%(90/355),28.2%(100/355),35.2%(125/355)and 22.5%(80/355),respectively.Additionally,mixed infection was observed.Meanwhile,849 samples of healthy pigs were tested by RT-PCR,and the results demonstrated that the total positive rate of PAstV was 8.13%(69/849).Of these,the proportion of PAstV-1,PAstV-2 and PAstV-4 infection was 27.5%(19/69),37.7%(26/69)and 40.6%(28/69),and a mixed infection also existed.Further sequencing and characterization of some the selected isolates revealed low sequence identities(56.2%)with known PAstV strains,indicating the presence of novel types or genotypes of PAstVs.Furthermore,the isolation conditions of porcine astrovirus were optimized,resulting in the purification of a pure PAstV-4 strain(designated PAstV-4-GRF1).The virus was found to exhibit typical astroviral morphology,with nucleotide identity ranging from 89.9 to 95.4%with previously published PAstV-4 strains.Then,macrovirus transcriptome sequencing showed that 88.30%of the CRF1 samples were mammalian astroviruses.By species classification,PAstV 4 and PAstV 2 accounted for 21.79%and 0.32%,respectively.Phylogenetic tree analysis showed that the c15050 fragment was identical to the GRF-1 sequencing fragment of the isolated strain,and exhibited the highest homology with the Hunan PAstV-4 sequence MK460231 in China.[Conclusion]As the inaugural isolated PAstV-4 strain,it furnishes pivotal materialfor the investigation ofthe biological and pathogenic properties of this virus as well as for the prospectivedevelopment of relevant biological and diagnostic reagents.
基金approved by the Ethical Committee for Human Subjects at Sir Run Run Shaw Hospital,Zhejiang University School of Medicine(20240276).All participants or their guardians provided written consent for their medical information to be used for publication.
文摘Objective Endoscopic tympanoplasty includes various surgical methods,such as internal repair,interlayer repair,and external overlay.This technique requires autologous materials,allografts,and xenografts,which are used to repair tympanic membrane(TM)perforation.To obtain good results,appropriate surgical methods and repair materials should be selected.This study aims to assess the efficacy of repairing refractory TM perforations in the porcine small intestinal submucosa(SIS)during transcanal endoscopic type I tympanoplasty.Method A retrospective chart review was performed on patients who underwent TM perforation repair with porcine SIS and tragus cartilage between January 2022 and September 2022 at Sir Run Run Shaw Hospital,Zhejiang University School of Medicine.Perforation size,tympanic status,pre-and postoperative symptoms,follow-up data,wound healing rates,and hearing improvement were analysed.Results Of the 115 patients included in the study,56 underwent interlayer repair with porcine SIS of the TM,and 59 patients underwent internal repair with tragus cartilage.No significant difference was found between the two groups at baseline in terms of age,sex,disease course,perforation side,tympanic status,underlying disease,or preoperative infection.The total postoperative effective rate of interlayer implantation with porcine SIS was 91.07%(51 patients),and that of internal implantation with tragus cartilage was 88.14%(52 patients).No significant difference was found in terms of the graft success rate between the two surgical methods(p=0.887).Postoperative pure tone auditory(PTA)and air-bone gap(ABG)density significantly increased in both groups compared with before surgery(p<0.05).However,the postoperative PTA and ABG density were not significantly different 3 months post-surgery between the two groups(p>0.05).Compared to those in the internal implantation group,the patients in the interlayer group had a shorter operation duration(51.36±6.76 min vs.59.71±7.45 min,t=6.298,p<0.001)and less blood loss(11.91±2.61 mL vs.15.27±2.57 mL,t=7.019,p<0.001).Conclusions Our study suggests that the porcine SIS,as well as the tragus cartilage,has a high success rate in repairing irreversible TM perforation.Endoscopic tympanoplasty via interlayer implantation with porcine SIS offers distinct advantages,including the absence of donor-site incision and scar formation,and ease of graft modification and manipulation.
文摘[ Objective] The aim of this study was to provide a theoretical basis for the prevention and treatment of highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS). [Method] Antigen location and histopathological observation in natural cases infected by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) were analyzed by immunohistochemistry and H. E. staining. [Result] The virus antigen mainly existed in epithelial calls, and also a few in mecrophages, lymphocytes and brain nerve cells. [ Conclusion] The cell and tissue tropism of HP-PRRSV strain in natural cases is different from that of previous strains.
基金Supported by Major Program of Natural Science Foundation of AnhuiProvince ( KJ2008A085)Anhui Key Technology R&D Program( 08010302179)~~
文摘To observe the acute toxicity of recombinant porcine interferen-alpha (IFN-alpha) in mice and thus provide a basis for the clinical safety. [Method] According to the principles of acute toxicity, all the mice were divided into two major groups (intraperitoneally injected group and intramuscularly injected group) respectively at high dose, moderate dose and low dose. And the normal control group was also set up. Within 14 d after administration, the behavior of mouse and the degree of toxicity were continuously observed. The hematological indexes and biochemical indexes of blood were detected to obtain the preliminary toxicity data of the recombinant porcine IFN-alpha. And at the end of the experiment, mice were sacrificed for autopsy. [ Result] There was not significant difference in external performance, behavioral characteristics, body temperature, weight, pathological anatomy of visceral organs, hematological indexes and biochemical indexes between the experimental groups and the control group. [ Conclusion] The highest dose of porcine interferon (5.0 x 10s IU per mouse) in this experiment or the dose lower than this dosage should not have significant toxic effects on mice, and the recombinant porcine IFN-alpha is safe in clinical application.
基金Supported by National Key Technology R&D Program (2006BAD06A12)Gansu Agricultural Biotechnology Research and Application Development Project (GNSW-2005-17)~~
文摘Three pairs of primer were designed for amplification of porcine pseudorabies virus (PRV) gB, gE, and TK gene by multiplex PCR (multi-PCR) in order to differentiate vaccine strains from field isolates. Three specific bands were obtained respectively at the expected size, 427 bp (gB gene), 298 bp (gEgene), and 208 bp (TKgene), Then four different gene-deleted vaccines of PRV were detected by multi-PCR. One ex- pected specific band was observed in one of samples, while two bands in the others. As shown by the detection results, the multi-PCR has high sensitivity and specificity and should be applied in pathogen diagnosis and epidemiological investigation in the future.
文摘[Objective] The aim of this study was to realize efficient expression of the porcine reproductive and respiratory syndrome virus (PRRSV) ORF7 gene in genetic engineering bacteria and analYze the immunological activity of the recombinant protein after purification. [ Method] The constructed recombinant expression vector pET-ORF7 was transformed into Escherichia co1BL21 (DE3) and induced by IPTG under the optimal condition. After analysis of SDS-PAGE and Western Blot, the expression products were purified by Ni-NTA His · Bind Resin chrom- atographic column under denaturing condition and renatured by gradient dialysis. Subsequently, the immunological activity of the renatured recombinant protein was detected by Westem Blot and indirect ELISA. [ Result] The recombinant plasmid pET-ORF7 expressed in E. coli successfully, and the fusion protein was in the form of inclusion body. By SDS-PAGE detection, the molecular weight of the expression protein was approximate 33 kD, according with the expectation. Analysis by Bandscan software showed that the expressed fusion protein was about 50% of total bacterial protein of BL21 (DE3). Wastem Blot and indirect ELISA detection showed that the renatured protein could react with PRRSV positive serum specifically, indicating its good immunological activity. [ Conclusion] This study lays a foundation for the preparation of PRRSV monoclonal antibody and diagnostic kit.
基金Supported by National High-tech Research and Development Plan (863 Project) in the Eleventh Five-Year Plan Period (2006AA10A207)~~
文摘[Objective] The purification and immunocompetence of GPS protein in porcine reproductive and respiratory syndrome (PRRS) were analyzed in this study, which provided basis for establishing the corresponding serological method. [Method] The recombinant expression plasmid pGEX-6P-5 was transformed into BL21 and expressed after being induced with IPTG. The solubility analysis of expression products was carried out, and then the recombinant protein was purified for SDS-PAGE identification and Western-blot analysis. Finally, the recombinant antigen was used in the immune experiment of guinea pigs. [Result] The target protein content accounted for 30% of the total cells protein content according to the chromatography scanning, and the purity of target protein after purification reached 80%. The purified protein was analyzed by Western-blot and immune experiment of guinea pigs, and the results showed that the expressed protein had good reactionogenicity and immunogenicity. [Conclusion] This study provides materials for further studies on the function between PRRSV ORF5 gene and its editing protein, which also lays a foundation for porcine reproductive and respiratory syndrome virus genetic engineering products.
基金Supported by 948 Project from Ministry of Agriculture(2006-G50)~~
文摘[ Objective] The aim of this study was to provide a basis for study on adiponectin as a candidate gene for fat deposition. [ Method] The promoter sequence of adiponectin was obtained by porcine BAC library screening and primer-walking method. The polymorphisms of adiponectin promoter from 290 pigs, including 5 breeds of Lantang pig, Large spotted pig, Large white pig, Landrace and Duroc, were analyzed with PCR-RFLP. [ Result] At SNP site of adiponectin 5'-flanking region -1 010 bp (G/A), GG genotype frequency in Chinese indigenous pigs was significantly higher than that in exotic pigs. At SNP site of adiponectin 5'-flanking region -394 bp (T/C), the genotype distribution of Chi- nese indigenous pigs was abundant, while no CC genotype was detected in exotic pigs, and T allele frequency was higher in exotic pigs. [ Conclusion] SNP site mutation of - 1 010 bp (G/A) may lead to changes of the gene transcription level, while SNP site of -394 bp (T/C) properly has no relationship with gene transcription level and fat deposition.
文摘[ Objective] The aim was to analyze the reason and epidemic trend of PRRSV, and provide theoretical basis for preventing and controlling PRRS. [Methed]According to the sequence of ATCC VR-2332 strain porcine reproductive and respiratory syndrome virus published by the GenBank, the primers were designed and synthesized. ORF5 gene sequences of seven prevalence strains were amplified by RT-PCR. The sequences of ORF5 genes were analyzed by DNAStar and compared with those of ATCC VR-2332, CH-1 a, B J-4, LV-M96262 and MLV vaccine strains, phylogenetic tree among isolates was analyzed. [Result] Analysis of nucleotide sequence showed that the homology was 88.1% - 98.8%, 89.9% -95.2%, 85.6% -98.7% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, BJ-4, and the homology was 54.7% -56.9% between ORF5 genes and LV. Analysis of amino acid sequence showed that the homology was 88.1% -96.8%, 88.1% - 94.5%, 86.1% -96.5% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, bBJ-4, the homology was 54.7% -56.2% between the ORF5 genes and LV.[ Conclusion] The variation of prevalence strains was great in the ORF5 gene region, the homology of ORF5 gene sequence was higher and genetic relationship was nearer during prevalence strains in the same region, or was far in different regions.
基金Supported by Project of Basic and Frontier Technology Research in Henan Province(072300430060 )Key Scientific and Technological Project of Henan Province(072102130023)~~
文摘[ Objective] To clone the porcine interleukin-18(/L-18) cDNA and explore the immunological effectiveness of porcine IL-18 as an adjuvant of genetic vaccine. [ Method] The spleen lymphccytes were isolated from Henan three-way cross-breeding pigs. According to the porcine IL-18 gene in GenBank, a pair of specific primers was designed. The full length cDNA of porcine IL-18 was amplified by RT-PCR. Subsequently, porcine IL-18 cDNA was cloned into pGEM-T vector and sequenced and analyzed. [ Result] The porcine IL-18 gene demonstrated an open reading frame of 579 bp encoding an inactive precursor protein with 192 amino acids. The precursor protein had no typical hydrophobic signal peptide and cleaved by interleukin-1 beta (IL-1β) converting enzyme(ICE) in caspase-1 splice site; the porcine mature protein had biological activity: After comparing with other porcine IL-18 genes, the nucleotide sequence homology was over 96% and the deduced amino acid homology was more than 98%. [ Conclusion] A full length procine IL-18 gene was gained. It lays the foundation for porcine IL-18 as an adjuvant of genetic vaccine.
文摘To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were then identified by immunohistochemical staining, and their culture viability measured through the MTT method in vitro. This induced them to differentiate into endocrine cells and detect their function. The isolated IPSCS did not express nestin, but expressed CK-19, a marker of ductal epithelia cells and ct-actin, a smooth muscle marker, demonstrating the growth characteristics of ES-like cells, and strong proliferative ability, after 18 passages. They could excrete insulin, and showed ultrastructure changes after being induced. Porcine pancreatic stem cells can be isolated by this method, induced to form islet-like clusters, and can secret insulin.
基金National High Technology Research and Development Program("863" Program) of China(No.2006AA02A132)
文摘AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.
基金Supported by the National Natural Science Foundation of China(No.81271716)
文摘AIM:To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix(APCM) in vitro.·METHODS:The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate(SDS)solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin(HE)staining and 4’,6-diamidino-2-phenylindole(DAPI)staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS:Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION:Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.
基金Supported by the Natural Science Foundation Program of Shandong Province(2007ZRA16001)~~
文摘[Objective] The research aimed to provide the theoretical basis for establishing a rapid diagnosis method for porcine parvovirus(PPV). [ Method] One pair of primers were designed according to PPV genome sequences on GenBank website and the sequences of prokaryotic expression vector pET30a ( + ) with multiple cloning sites. The whole sequence of NS1 gene in PPV SD1 strain was amplified by using PCR technology and the positive recombinant plasmid was analyzed by sequencing and homology comparison. The prokaryotic expression recombinant plasmid PET30a/NS1 was constructed to make its induction expression in Escherichia coll. [ Result] The target fragment with the length of 2 208 bp was obtained from PCR amplification. The nucleotide homologies between the cloned NS1 gene and the reported relevant PPV genes were from 97.3 % to 99.4 %, which indicated that NS1 gene had high conservation. But it had a 12-basepair successive deletion near the hydroxyl end. The cloned PPV NS1 gene was successfully expressed in prokaryotic cell, and its expression products existed mostly in inclusion bodies. [ Conclusion] The results of SDS-PAGE detection showed that the molecular weight of PPV NS1 protein was 86 KD.
基金Supported by 863 Program of China(2007AA10Z161)National Natural Science Foundation of China(30771545)~~
文摘[Objective]The aim was to study the differential expression ofAPOEgene in different generations of porcine fetal fibroblasts cells.[Method]The first,tenth,fifteenth,twentieth,twenty-fifth,fiftieth generations of porcine fetal fibroblast cells,which were normally grown and passed,were collected before total RNA was extracted respectively.The expression ofAPOEgene in different generations of porcine fetal fibro-blast cells was detected by RT-PCR technique.[Result]The expression level of porcine APOE mRNA in the first generation of porcine fetal fi-broblast cells was the highest,and then it gradually decreased with the increase of cell generations and was the lowest in the fiftieth generation.[Conclusion]The expression ofAPOEgene had the selective trend in different generations of porcine fetal fibroblast cells.
基金supported by grants from the National Basic Research Program of China (973 Program,2005CB523200)the National High-Tech Research and Development Program of China (863 Program,2006AA10A20 4)+1 种基金the National Key Technology R&D Program (2006BAD 06A04/18/01/03)the National Natural Science Foundation of China (30470072)
文摘Since May 2006,a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant characterized by 30 amino acids deletion within its NSP2-coding region emerged and caused extensive economic losses to China's pig industry.To investigate the in vivo pathogenicity and immune responses of the newly emerging PRRSV,3 groups of 60-d-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant HuN4 with 3 different infection doses (3×103-3×105 TCID50).The results revealed that the virus variant caused severe disease in piglets and the significant clinical characteristics consisted of persistently high fever (41.0-41.9oC) and high morbidity and mortality (60-100%),the marked clinical signs of PRRS and severe histopathogenic damages in multiple organs.It induced rapid and intense humoral immune responses and seroconversion was detected in most infected pigs at 7 d post-infection (DPI).The virus vigorously replicated in vivo and the highest virus average titer was 9.7 log copies mL-1 serum at 7 DPI.Elevated levels of IFN-g and IL-10 cytokine production in serum in this study were also observed.Taken together,our results demonstrated that the HP-PRRSV variant HuN4 strain is highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines.