Recombinant chitinase-3-like protein 1 alleviates learning and memory impairments via M2 microglia polarization in postoperative cognitive dysfunction mice

Postoperative cognitive dysfunction is a seve re complication of the central nervous system that occurs after anesthesia and surgery,and has received attention for its high incidence and effect on the quality of life of patients.To date,there are no viable treatment options for postoperative cognitive dysfunction.The identification of postoperative cognitive dysfunction hub genes could provide new research directions and therapeutic targets for future research.To identify the signaling mechanisms contributing to postoperative cognitive dysfunction,we first conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the Gene Expression Omnibus GSE95426 dataset,which consists of mRNAs and long non-coding RNAs differentially expressed in mouse hippocampus3 days after tibial fracture.The dataset was enriched in genes associated with the biological process"regulation of immune cells,"of which Chill was identified as a hub gene.Therefore,we investigated the contribution of chitinase-3-like protein 1 protein expression changes to postoperative cognitive dysfunction in the mouse model of tibial fractu re surgery.Mice were intraperitoneally injected with vehicle or recombinant chitinase-3-like protein 124 hours post-surgery,and the injection groups were compared with untreated control mice for learning and memory capacities using the Y-maze and fear conditioning tests.In addition,protein expression levels of proinflammatory factors(interleukin-1βand inducible nitric oxide synthase),M2-type macrophage markers(CD206 and arginase-1),and cognition-related proteins(brain-derived neurotropic factor and phosphorylated NMDA receptor subunit NR2B)were measured in hippocampus by western blotting.Treatment with recombinant chitinase-3-like protein 1 prevented surgery-induced cognitive impairment,downregulated interleukin-1βand nducible nitric oxide synthase expression,and upregulated CD206,arginase-1,pNR2B,and brain-derived neurotropic factor expression compared with vehicle treatment.Intraperitoneal administration of the specific ERK inhibitor PD98059 diminished the effects of recombinant chitinase-3-like protein 1.Collectively,our findings suggest that recombinant chitinase-3-like protein 1 ameliorates surgery-induced cognitive decline by attenuating neuroinflammation via M2 microglial polarization in the hippocampus.Therefore,recombinant chitinase-3-like protein1 may have therapeutic potential fo r postoperative cognitive dysfunction.

EFFECT OF RECOMBINANT MOUSE INTERLEUKIN-3 ON HEMATOPOIESIS IN NORMAL AND CYCLOPHOSPHAMIDE-TREATED MICE

Intraperitoneal injection of recombinant mouse IL-3 in normal mice for 6 days did not induce any significant changes in leukocyte and neutrophil counts. In comparison with saline controls, IL-3-treated mice experienced a 1.7-fold increase of bone marrow nucleated cells and 3.6-fold increase of CFU-GM colonies. Tritium thymidine incorporation of bone marrow cells significantly increased in IL-3-treated mice as compared with that in normal saline-treated mice. These results suggested that IL-3 expanded the number of granulocyte-macrophage progenitor cells but not the peripheral neutrophils. We examined the effect of IL-3 on hematopoietic reconstitution in a cyclophosphamide-treated mouse model. We found that the absolute neutrophil number of mice treated with IL-3 increased significantly on day 6 post injection when compared with the control mice (6.2± 4.1×109 / L versus 0.98± 1.4 × 109/ L, P【0.01) . The results demonstrated that IL-3 stimulated an early recovery of neutrophils after

Recombinant adenovirus-p53(Gendicine) sensitizes a pancreatic carcinoma cell line to radiation

Objective： In this study, we examine the effects of recombinant adenovirus-p53 （rAd-p53） on the pancreatic carcinoma cell line SW1990. Specifically, we determine if expression of rAd-p53 sensitizes these cells to radiation. Methods： Following transfection of SW1990 cells with rAd-p53, we measured expression of P53, P21 and Bax by immunocytochemistry. Both transfected and control cell lines were irradiated with a range of doses, and the survival fractions （SF） were calculated. Dose survival cttrves were constructed and modeled for comparison. Results： Transfection of SW1990 cells with rAd-p53 resulted in increased expression of P53, P21 and Bax in a time-dependent manner. At 96 h after transfection, 89.92% of cells expressed P53, 56.8% expressed P21, and 76.50% expressed Bax. The SF following radiation was lower in the rAd-p53 transfected cells compared to the control cells, suggesting that rAd-p53 sensitizes SW1990 cells to radiation （Do for the experimental and control groups was 2.199 and 2.462, respectively）. Conclusions： Use of the adenoviral vector is an effective means of transfecting SW1990 cells with wild-type P53, and this sensitizes the cell line to irradiation. This work suggests that combining rAd-p53 with radiation therapy in pancreatic cancer may be therapeutically beneficial.

Study of recombinant human interleukin-12 for treatment of complications after radiotherapy for tumor patients

AIM To evaluate the treatment effects of recombinant human interleukin-12(rh IL-12) on radiotherapy complications, such as severe myelosuppression or pancytopenia, the decline or imbalance of immune function, etc.METHODS The patients received high-dose and short-course precise radiotherapy, such as Cyber knife and image-guided radiotherapy(IGRT), which can cause myelosuppression or pancytopenia and immune function decline within a short time. One-hundred subjects were enrolled in the study, and 50 were randomized to a treatment group which used rh IL-12 and 50 were randomized to a control group which used symptomatic and supportive therapy after radiotherapy. The 50 subjects in the treatment group were further divided into five subgroups and intervenedwith rh IL-12 at a dose of 50, 100, 150, 200 or 250 ng/kg respectively. The dose-effect relationship was observed. RESULTS Rh IL-12 significantly attenuated the decrease of peripheral blood cells in the treatment group, and immune function was improved after treatment. Due to the different radiation doses, there was a fluctuation within 12 h after treatment but mostly showing an increasing trend. As to the clinical manifestations, 2 patients in the 250 ng/kg subgroup showed low fever after administration, 1 patient in the 200 ng/kg subgroup and 2 patients in the 250 ng/kg subgroup showed mild impairment of liver function during the observation period.CONCLUSION Rh IL-12 has effective therapeutic and protective effects on complications following radiotherapy, such as the decline of blood cells, myelosuppression and the decline or imbalance of immune function, which indicated good prospects for development and application.

Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

BACKGROUND： Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. OBJECTIVE： To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. DESIGN： Gene directed cloning. SETTING： Central Laboratory of Northern China Coal Medical College. MATERIALS： DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. METHODS： NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid （pAAV-NT-3）. pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10：1 and the mixture was used to infect HT 1080 cells. X-gal stain was used to measure virus titer. MAIN OUTCOME MEASURES： Size of targeted gene fragments, validity of vehicle construction and virus titer. RESULTS： Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells （HT1080） and the recombinant virus titer was measured as 1 ×10^12 virus particles per milliliter. CONCLUSION： Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.

Recombinant human interleukin-11 for treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer

Objective： To evaluate the efficacy and safety of recombinant human interleukin-11 （rhIL-11） for the chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer. Methods： It was an opened and non-randomized controlled clinical study. When the platelet counts was under 75 × 10^9/L after chemotherapy, rhlL-11 was administered 25 μg/（kg·d） as a daily SC injection last for 7-14 days, or discontinued when platelet counts 〉 100 × 10^9/L. Results： Seventysix patients were enrolled into this study. The treatment group and the control group had thirty-eight cases, respectively. The mean recovery time to PLT ≥ 100 × 10^9/L was 8.1 days in treatment group, while in control group was 12.2 days （P 〈 0.01）. Moreover, the mean recovery time from PLT 〈_ 50 × 10^9/L to 〉 100 × 10^9/L was 8.9 days in treatment group, while in control group was 12.9 days （P 〈 0.05）. There was a statistical difference between the two groups. Major side effects included edema, fever, articular muscle soreness, but they were all mild and well tolerable. Conclusion： rhIL-11 can be safely and effectively used for the treatment of chemotherapy-induced thrombocytopenia in patients with gastrointestinal cancer.

Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved.Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process.AIM To assess the influence of interleukin-10(IL-10)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)following their interaction with macrophages in an inflammatory environment.METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment.In this study,we investigated its impact on the proliferation,migration,and osteogenesis of BMSCs.The expression levels of signal transducer and activator of transcription 3(STAT3)and its activated form,phos-phorylated-STAT3,were examined in IL-10-stimulated macrophages.Subsequently,a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling.RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution,and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs.Mechanistically,STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages.Specifically,IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response,as evidenced by its diminished impact on the osteogenic differentiation of BMSCs.CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs.The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’osteogenic differentiation.

Effect of artmether,hemin and Fe^(3+) on recombinant lactate dehydrogenase from Schistosoma japonicum

Objective:To explore antischitosome effects of artemether,hemin and Fe on S/LDH.Methods: Enzyme activity of rS/LDH was assayed in the standard reaction system by adding different concentration of reagents(0.00-0.10 mM artemether,0.00-0.02 mM hemin,0.00-0.50 mM Fe^(3+)). Same solvents of the each reagent were used as control.Results:There was no enzyme activity inhibition observed at 0.10 mM artemther:obivious inhibition for lactate oxidation reaction and pyruvate reduction reaction were detected at 0.002 mM and 0.004 mM of hemin,respectively: comparing with that of the control(P<0.05).The relative enzymatic activity inhibitions for pyruvate reduction reaction and lactate oxidation reaction at 0.02 mM hemin were 93.48%and 100.00%,respectively,comparing with that of the control(P<0.01):both pyruvate reduction and lactate oxidation reaction were inhibited completely at 0.50 mM Fe^(3+),comparing with that of the control(P<0.01).Conclusions:The results implied that SjLDH was not the direct molecular target of artemether.Hemin and Fe are inhibitors of SjLDH.

Induction of Chondrogenesis of Adipose-derived Stem Cells by Novel Recombinant TGF-β3 Fusion Protein

Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.

Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase

3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human （h） and rat （r）. Recombinant Ad-r（h）SYN1-DHCR24 was transfected into AD-293, N2A （mouse neuroblastoma）, and MIN6 （mouse pancreatic carcinoma） cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer＇s disease.

Neuronal changes in the retinal ganglion cell layer following recombinant human interleukin-2 intravitreal injection in a rat model of chronically elevated intraocular pressure

Intraperitoneal injection of recombinant human interleukin-2（rhIL-2）inhibits neuronal apoptosis in the chronic ocular hypertension retinal ganglion cell layer.Intravitreous injection was performed on retinal ganglion cells in a Wistar rat model of chronically elevated intraocular pressure to observe the effects of LY294002 and AG490 on retinal ganglion cell survival,macrophage activation,and PI3K/Akt and JAK/STAT activation.The number of retinal ganglion cells in the rhIL-2 treatment group was much greater than in the normal control and phosphate-buffered saline groups.Western blot analysis revealed low Akt and STAT3 protein expression in the retina after 3-hour intravitreous injections of rhIL-2.However,protein expression was increased at 12 hours,but decreased again at 24 hours,with very low expression at 96 hours.LY294002 and AG490,which are inhibitors of the PI3K/Akt and JAK/STAT3 signal pathways,prevented upregulation of Akt and STAT3 protein expression in the retina,respectively.Intravitreous injection of rhIL-2 exhibited neuroprotective effects by decreasing retinal ganglion cell layer damage in a rat model of chronic glaucoma.These results suggest that intravitreal injection of rhIL-2 could induce the PI3K/Akt and JAK/STAT3 signaling pathways to protect retinal ganglion cells in chronically elevated intraocular pressure models.

Detection of Antibodies against Toxoplasma gondii by ELISA with Recombinant Microneme Protein 3

[Objective] To develop a new method for serodiagnosis of swine toxoplasmosis. [Method] With the purified recombinant microneme protein 3 （rMIC3） as coating antigens, an indirect ELISA was developed for detection of antibodies against Toxoplasma gondii. [ Result] The optimal working concentration of rMIC3 was 3. 40 ug/ml, and the optimal degree of dilution of sera was 1：160. Cross-reaction was not observed between the Toxoplasma gondii-positive sera and the positive sera against classical swine fever virus or some other pathogens. The developed ELISA had 92.56% coincidence rate with latex agglutination test. [ Conclusion] The developed ELISA is sensitive, rapid, specific and reproducible, and thus it can be applied in serodiagnosis and seroprevalence investigation of swine toxoplasmosis.

Anti-VDAC3 recombinant antibody decreased human sperm motility and membrane integrity: A potential spermicide for contraception

Objective:To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded.Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly.Conclusions:Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

Expression and Characterization of the Recombinant Human FLT-3 Ligand Extracellular Domain in Pichia Pastoris

OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris （P. pastoris）strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5＇AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34＋cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield （108 mg/L） was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34＋ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34＋ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.

Optimization for Purification and Characterization of Recombinant Hirudin Ⅲ from E. coli

Aim To optimize purification conditions of recombinant hirudin 3 in thefermentation broth and characterize the product. Methods Reambinant hirudin 3 was isolated andpurified from the fermentation broth by three column chromatography steps with macroporous resin,DEAE cellulose DES2 and preparative RP-HPLC, respectively, and the optimal conditions were obtained.Purity of the product was determined by SDS-PAGE and analytical RP-HPLC. The molecular weight wasdetermined by mass spec-trometry. The structure of the product was analyzed by peptide map.ResultsThe product with purity of 95.4786% was obtained after three purification steps in the optimumconditions with a total yield of 39%. The molecular weight of the product was 6 913.32 ± 6.55 Da,coincident to the theoretical molecular weight of r-hirudin 3. The structure of the product wascoincident to r-hirudin 3 either. Conclusion The optimized purification steps can be successfullyemployed for purification of r-hirudin 3 from E. coli using batch-type approaches. The productobtained with high purity was confirmed to be r-hirudin 3.

泄浊养血法联合重组人促红素注射液治疗肾虚湿浊型慢性肾脏病3~5期肾性贫血患者的临床观察

[目的]观察泄浊养血法联合重组人促红素注射液对慢性肾脏病(CKD)3~5期肾性贫血患者贫血改善及残余肾功能的干预作用。[方法]选择2020年10月—2021年10月就诊于天津中医药大学第一附属医院的88例CKD 3~5期肾性贫血患者,根据随机数字表法随机分为对照组(44例)和治疗组(44例)。对照组予重组人促红细胞生成素注射液及多糖铁复合物治疗,治疗组在此基础上联合泄浊养血法中药方治疗,连续服用3个月,观察治疗前后两组临床疗效、红细胞计数(RBC)、血红蛋白(Hb)、血清肌酐(Scr)、尿素氮(BUN)、肾小球滤过率(eGFR)、尿微量白蛋白(mALB)、丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)的变化,症状积分及不良反应发生率。[结果]治疗3个月后,治疗组总有效率为86.36%,对照组为56.82%,治疗组临床疗效优于对照组(P<0.05);症状积分、RBC、Hb、Scr、BUN、mALB均较治疗前改善,且治疗组优于对照组(P<0.05);安全性指标中AST、ALT治疗前后数值变化,差异无统计学意义(P>0.05)。两组病例中均未出现不良反应。[结论]泄浊养血法联合重组人促红细胞生成素注射液治疗可以改善CKD 3~5期非透析肾性贫血患者临床症状,提高临床疗效,延缓肾功能进展,且具有一定的安全性。

鸭甲型肝炎病毒3型信阳株的分离鉴定及遗传演化分析

为了解信阳地区鸭甲型肝炎病毒3型的遗传变异特性,对信阳市发生疑似肝炎的金定雏鸭进行9种常见病毒PCR/RT-PCR检测,同时采集病鸭肝脏组织无菌处理后,尿囊腔接种10日龄SPF鸭胚进行病毒分离,并对分离毒株进行全基因组和VP1基因序列分析。结果:成功分离到1株鸭甲型肝炎病毒3型(DHAV3),命名为HNXY23;接种病毒的鸭胚发育不良,死亡胚体全身呈出血水肿;序列比对和系统发育树表明,分离株HNXY23属于DHAV3 GⅠ基因型,与2022年分离株HB-1、HZ-1、HZ-2和HZ-3亲缘关系较近;利用DNAMAN软件比对31株DHAV3 VP1氨基酸位点变异情况,发现在184、187、195、206和209位这5个氨基酸位点有较为明显的变化差异。本研究结果丰富了信阳地区DHAV3的分子流行病学资料,为进一步研究DHAV3的致病机制奠定了基础。

Expression of interleukin-22/STAT3 signaling pathway in ulcerative colitis and related carcinogenesis

AIM:To investigate the expression of interleukin (IL)-22 and its related proteins in biopsy specimens from patients with ulcerative colitis (UC) and UC-related carcinogenesis. METHODS:Biopsy specimens were obtained from patients with inactive (n = 10), mild-to-moderately active (n = 30), severely active (n = 34), initial (n = 30), and chronic UC (n = 44), as well as UC patients with dysplasia (n = 10). Specimens from patients without colonic abnormalities (n = 20) served as controls. Chronic colitis in experimental mice was induced by 2.5% dextran sodium sulfate. The expression levels of IL-22, IL-23, IL-22R1 and phosphorylated STAT3 (p- STAT3) were determined by immunohistochemistry. Bcl-2, cyclin D1 and survivin expression was detected by Western blotting. RESULTS:Patients with active UC had significantly more IL-22, IL-23, IL-22R1 and p-STAT3-positive cells than the patients with inactive UC and normal controls. Furthermore, IL-22 and related proteins were closely related to the severity of the colitis. The expression of IL-22 and IL-22R1 in the tissue of initial UC was stronger than in that of chronic UC, whereas the expression of p-STAT3 was significantly increased in chronic UC tissues. In dysplasia tissues, the expression level of IL-22 and related proteins was higher compared with controls. Mouse colitis model showed that expression of IL-22, IL-22R1 and IL-23 was increased with time, p-STAT3 and the downstream gene were also remarkably upregulated.CONCLUSION:IL-22/STAT3 signaling pathway may be related to UC and UC-induced carcinogenesis and IL-22 can be used as a biomarker in judging the severity of UC.

The Expression of Interleukin-17, Interferon-gamma, and Macrophage Inflammatory Protein-3 Alpha mRNA in Patients with Psoriasis Vulgaris

Summary: To investigate the role of Interleukin-17 (IL-17), Interferon-gamma (IFN-γ), and macrophage inflammatory protein-3 alpha (MIP-3α) in the pathogenesis of psoriasis, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to semi-quantitatively analyze the mRNA expression of IL-17, IFN-γ, and MIP-3α in 31 psoriatic lesions and 16 normal skin tissues. The results showed that the mRNA of the three cytokines was present in all specimens. And the expression level of IL-17 mRNA in skin lesions was 1.1416±0.0591, which was significantly higher than that in normal controls (0.8788±0.0344, P<0.001). The expression levels of IFN-γ mRNA were 1.1142±0.0561 and 0.9050±0.0263, respectively, with significant difference(P<0.001). And the expression levels of MIP-3α mRNA in psoriatic lesions was 1.1397±0.0521, which was markedly higher than that in normal controls (0.8681±0.0308, P<0.001). These findings indicate that up-regulated expression of IL-17, IFN-γ, and MIP-3α might be involved in the pathogenesis of psoriasis.

Metabolic syndrome attenuates ulcerative colitis: Correlation with interleukin-10 and galectin-3 expression

BACKGROUND Ulcerative colitis(UC)is a chronic disease characterized by inflammation of intestinal epithelium,primarily of the colon.An increasing prevalence of metabolic syndrome(MetS)in patients with UC has been documented recently.Still,there is no evidence that MetS alters the course of the UC.AIM To test the influence of the MetS on the severity of UC and the local and systemic immune status.METHODS Eighty nine patients with de novo histologically confirmed UC were divided in two groups,according to ATP III criteria:Group without MetS(no MetS)and group with MetS.RESULTS Clinically and histologically milder disease with higher serum level of immunosuppressive cytokine interleukin-10(IL-10)and fecal content of Galectin-3(Gal-3)was observed in subjects with UC and MetS,compared to subjects suffering from UC only.This was accompanied with predomination of IL-10 over pro-inflammatory cytokines tumor necrosis factorα(TNF-α),interleukin-6(IL-6),and interleukin-17(IL-17)in the sera as well as Gal-3 over TNF-αand IL-17 in feces of UC patients with MetS.Further,the patients with both conditions(UC and MetS)had higher percentage of IL-10 producing and Gal-3 expressing innate and acquired immune cells in lamina propria.CONCLUSION Local dominance of Gal-3 and IL-10 over pro-inflammatory mediators in patients with MetS may present a mechanism for limiting the inflammatory process and subsequent tissue damage in UC.