HIGH EFFICIENCY RETROVIRUS-MEDIATED GENE TRANSFERTO LEUKEMIA CELLS

Objective: To establish an efficient and safe gene transfer system mediated by retrovirus for gene marking and gene therapy of human leukemia. Method: The retroviral vector LXSN, containing the neomycin resistance (NeoR) gene, was transferred into amphotropic packaging cells GP+envAm12 by liposome transfection or by ecotropic retrovirus transduction. Amphotropic retrovirus in supernatants with higher titer was used to infect human leukemic cell lines NB4, U937, and THP-1. The efficiency of gene transfer was assayed on colonies formed by transduced K562 cells. Results: The titer of DOSPER directly transfected GP+envAm12 cells determined on NIH3T3 cells was 8.0×105 CFU/ml, while that of producer infected with retrovirus was 1.6×107 CFU/ml. Integration of NeoR gene into all leukemia cells was confirmed by polymerase chain reaction (PCR). Absence of replication-competent virus was proved by both nested PCR for env gene and marker gene rescue assay. Gene transfer with the efficiency as high as 93.3 to 100% in K562 cells was verified by seminested PCR for integrated NeoR gene on colonies after 7 days’ culture. Conclusion: The efficiency and safety of retrovirus mediated gene transfer system might provide an optimal system in gene therapy for leukemia or genetic diseases.

Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells

Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.

Study of the "killing" effect of retrovirus-mediated HyTK gene transfer on melanoma cell line

Recent research showed that the transfer of the Herpes simplex virus thymidine kinase (HSV tk) gene into malignant tumor cells could confer the tumor cells susceptibility to the antiviral drug ganciclovir (GCY), thus produce "killing" effect selectively on the tumor cells exposed to GCV. We constructed a recombinant retroviral vector LHyTK/N by inserting HyTK gene into the retroviral vector LXSN and cutting out the SV4O early promoter. The HyTK gene was transferred into mouse melanoma cell line B16 mediated by a recombinant virus. PCR analysis showed that the HyTK gene was successfully transferred and replication-competent virus was absent. The "killing" effect on B16/HyTK+ cells exposed to GCV (>0. 1 μmol/L) was evident when investigated under light microscope and by live cell counting.

Experimental study on ex vivo retrovirus-mediated aFGF gene transfer therapy in traumatic brain injury

Objective:To exploretheeffectsof ex vivo retrovirus-mediatedgenetransfertherapywithacidicfibroblast growthfactor(aFGF)in themanagementof traumaticbraininjury.Methods:PLXSN-SPaFGF,a recombinantretroviral vectorexpressingbiologicallyactiveaFGFwas constructedandtransfectedintoculturedembryonicastroglialcellswhich wereinjectedintothesurroundingareasof thecontusionin theratleftparietalcortex.From3d to1monthaftertheim-plantation,thesurvivalof andaFGFgeneexpressionintheimplantedastroglialcellswereexamined,andneuronalapopto-sisandratmotorfunctionimpairmentevaluated.Re sults:TheimplantedaFGF-transducedastroglialcellssurvivedandex-pressedaFGFmRNAandproteinevidentlyat3d aftergrafting.Thenumberof andaFGFgeneexpressionintheastroglial cellsincreasedremarkebly7d anddecreasedto someextent1monthaftertheimplantation.ThereweresignificantaFGF mRNA andproteinexpressionin theneuronssurroundingthecontusionat7d thatdecreasedto relativelylow levels1monthaftertheimplantationof aFGF-transduedastrocytes.Diminishedneuronalapoptosis(P<0.05)andsignificantlyim-provedinthepreviouslyimpairedmotorfunction(P<0.05)of theratswereobservedfrom7d to1monthaftertheimplan-tation.Con clu sion:Thisexperimentsuccessfullyconductedex vivo aFGFgenetransfertherapyin traumaticbraininjury whichprovedto be effectiveinrescuinginjurednervecellfromdeathandenhancingrecoveryof neurologicaldeficiency.

RETROVIRUS-MEDIATED TNF-α GENE TRANSFER INTO TCA8113 CELLS

Objective To investigate whether TNF-α gene-modified Tca8113 cells (Tca8113/TNF-α) canbe used as vaccine for oral squamous cell carcinoma. Methods TNF-α gene was transduced into Tca8113 cellsin vitro with retroviral vector carring genes for both TNF-α and NeoR. After that, presence and expression of exoge-nous gene in the transgenic cells, expression of HLA antigen on the cells, expression of TNF-α and survival rate ofthe cells after irradiation and cryopreservation, and mutagenic activity of the cells were analyzed by PCR technique,EL1SA technique, FACS technique, 60Co irradiation inactivation test, cryopreservation test, and Ames test, respec-tively. Results The presence of both TNF-a and NeoR gene and expression of TNF-α gene were demonstrated intransgenic cells. The levels of the HLA-A, B, C, DR expressed by Tca8113/TNF-α were higher than by the parentalcells. Tca8113/TNF-α continued to secrete TNF-α for 14 d, there was a secretion peak time from d4 to d6;and, allthe cells died by dl4 after irradiation. The Level of TNF-α secreted by Tca8113/TNF-α cryopreserved for 48 h wasno different from that cryopreserved for 1 week after irradiation, the level of TNF-α secreted by the cryopreservedcells was just a little lower than that secreted by the noncryopreserved cells. Both DNA and supernatant of the cellshave no mutagenic activity. Conclusion TNF-α gene can be transduced into Tca8113 cells with retroviral vec-tor, and the cells can express TNF-α. Expression of HLA 1,11 antigens on Tca8113 cells can be increased by TNF-αgene transduction. Irradiation is a reliable inactivation method, and cryopreservation is a feasible conservationmethod for Tca8113/TNF-α. Ames test result indicate that Tca8113/TNF-α has no mutagenic activity.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

Construction and Identification of a Goat Pox Virus Transfer Vector to Express Peste des Petits Ruminants H gene

[Objective] This study was to develop a live vector vaccine of goat pox virus of Peste des petits ruminants(PPR). [Method] Using PCR amplification technique, PPR H gene was obtained, then ligated into pGEM-T easy vector; the recombinants were digested by Nhe Ⅰ and Hind Ⅲ, and ligated into pEGFP-N1-P7.5, yielding the recombinant vector pEGFP-N1-P7.5-H; next the expression cassette EGFP-N1-P7.5-H was first released from recombinant vector pEGFP-N1-P7.5-H by double digestion of Hind Ⅲ and Nhe Ⅰ and ligated into pUC119-TK that was digested by Kpn Ⅰ, yielding the transfer vector pUC119-TK-EGFP-P7.5-H. [Result] Identification and double enzyme digestion showed that the transfer vector pUC119-TK-EGFP-P7.5-H was correctly constructed. From the transfer vector transfected BHK-21 cells which infected GTPV AV41, specific fluorescence was observed at 48th h of transfection. [Conclusion] The construction of goat poxvirus live vector laid a foundation for the live vector vaccine of PPR vaccine.

cGH Gene Transfer and PCR Detection in Crucian Carp

By inserting the CMV with HSV TK promoter into the plasmid pBlue-cGH which contained cGH gene, a chimeric expression vector was constructed for transgenic fish. After the vector was transferred into fertilized eggs of crucian carp by means of microinjection, the transgenic crucian carp was obtained. The result of PCR detection showed that the growth hormone gene of the exogenous carp was integrated into the genome of crucian carp, and the transgenic crucian carp displayed enhancement of its growth.

Gene therapy for human colorectal carcinoma using human CEA promoter controlled bacterial ADP-ribosylating toxin genes:PEA and DTA gene transfer

AIM To establish a tissue-specific gene therapy for colorectal carcinoma using bacterial ADP-ribosylating toxin genes.METHODS Pseudomonas exotoxin A domain Ⅱ+Ⅲ (PEA) was cloned from genomic DNA of Pseudomonas aeruginosa. PEA and diphtheria toxin A chain gene (DTA) were modified to express eukaryotically. After sequencing, the toxin genes under the control of human carcinoembryonic antigen (CEA) promoter were cloned into retroviral vectors to construct CEAPEA and CEADTA respectively. In vitro cotransfection of the constructs with luciferase vectors and in vivo gene transfer in nude mice were subsequently carried out.RESULTS Both CEAPEA and CEADTA specifically inhibited the reporter gene expression in the CEA positive human colorectal carcinoma (CRC) cells in vitro. Direct injection of CEAPEA and CEADTA constructs into the established human tumors in BALB/c nude mice led to significant and selective reductions in CRC tumor size as compared with that in control groups.CONCLUSION The toxin genes, working as therapeutic genes, are suitable for the tissue-specific gene therapy for colorectal carcinoma.

RETROVIRAL MEDIATED EFFICIENT TRANSFER ANDEXPRESSION OF MULTIPLE DRUG RESISTANCE GENE TO HUMAN LEUKEMIC CELLS

Objective: To investigate retroviral-mediated transfer and expression of human multidrug resistance (MDR) gene MDR1 in leukemic cells. Methods: Human myeloid cells, K562 and NB4, were infected by MDR retrovirus from the producer PA317/HaMDR, and the resistant cells were selected with cytotoxic drug. The transfer and expression of MDR1 gene was analyzed by using polymerase chain reaction (PCR), flow cytometry (FCM) and semisolid colonies cultivation. Results: The resistant cells, K562/MDR and NB4/MDR, in which integration of the exogenous MDR1 gene was confirmed by PCR analysis, displayed a typical MDR phenotype. The expression of MDR1 transgene was detected on truncated as well as full-length transcripts. Moreover, the resistant cells were P-glycoprotein postiive at 78.0% to 98.7% analyzed with FCM. The transduction efficieny in K562 cells was studied on suspension cultures and single-cell colonies. The transduction was more efficient in coculture system (67.9%-72.5%) than in supernatant system (33.1%~46.8%), while growth factors may improve the efficiency. Conclusion: Retrovirus could allow a functional transfer and expression of MDR1 gene in human leukemia cells, and MDR1 might act as a dominant selectable gene for coexpression with the genes of interest in gene therapy.

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer （MGCMGT） or female germ cell mediated gene transfer （FGCMGT） technique. Sperm-mediated gene transfer （SMGT）, including its alternative method, testis-mediated gene transfer （TMGT）, becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer （OMGT） is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.

Adenoviral-mediated Hath1-EGFP gene transfer into guinea pig cochlea through intact round window membrane

Objective To study expression of adenoviral-mediated Hath1-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane(RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with Ad-Hath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and frozen sections were examined. Results ABR tests showed no significant change of hearing after the surgery. Strong fluorescence staining in the cochleae was seen in Ad-Hath1-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with little decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.

Adenovirus-mediated FasL gene transfer into human gastric carcinoma

AIM: To evaluate the possible value of FasL in gastric cancer gene therapy by investigating the effects of FasL expression on human gastric cancer cell line. METHODS: An adenoviral vector encoding the full-length human FasL cDNA was constructed and used to infect a human gastric cancer (SGC-7901) cell line. FasL expression was confirmed by X-gal staining, flow cytometric analysis and RT-PCR. The effect of FasL on cell proliferation was determined by clonogenic assay, cytotoxicity was detected by MTT assay, and cell viability was measured by trypan blue exclusion. The therapeutic efficiency of Ad-FasL in vivo was investigated with a xenograft tumor model in nude mice. RESULTS: SGC-7901 cells infected with Ad-FasL showed increased expression of FasL, resulting in significantly decreased cell growth and colony-forming activity when compared with control adenovirus-infected cells. The cytotoxicity of anti-Fas antibody (CH-11) in gastric cancer cells was stronger than that of ActD (91±8 vs60±5,P<0.01), and the cytotoxicity of Ad-FasL was stronger than that of CH-11 (60±5 vs50±2, P<0.05). In addition, G1-phase arrest (67.75±0.39 vs 58.03±2.16, P<0.05) and apoptosis were observed in Ad-FasL-infected SGC-7901 cells, and the growth of SGC-7901 xenografts in nude mice was retarded after intra-tumoral injection with Ad-FasL (54% vs 0%,P<0.0001). CONCLUSION: Infection of human gastric carcinoma cells with Ad-FasL induces apoptosis, indicating that this target gene might be of potential value in gene therapy for gastric cancer.

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Erythropoietin gene transfer into rat testes by in vivo electropo-ration may reduce the risk of germ cell loss caused by cryptorchidism

Aim： To investigate the effects of rat Erythropoietin （Epo） on spermatogenesis by transferring rat Epo gene into cryptorchid testes by means of in vivo electroporation. Methods： Sprague-Dawley rats with surgically-induced unilateral cryptorchidism were divided into three groups： the first group was given intratesticular injections of pCAGGS-Epo （pCAGGS-Epo group）, the second group was given intratesticular injections of pCAGGS （pCAGGS group）, and the third group were given intratesticular injections of phosphate-buffered saline （PBS group）. At the same time, square electric pulses of 30 V were applied six times with a time constant of 100 ms. One or two weeks after injection, each testis was weighed and the ratio of the total number of germ cells to that of Sertoli cells （G/S ratio） was calculated to evaluate the impairment of spermatogenesis. Ten testes taken from each of the three groups were examined at each time point. Results： The testicular weight after the injection of pCAGGS-Epo or pCAGGS control plasmid was （0.85 ± 0.08） g and （0.83 ± 0.03） g, respectively, at week 1 （P = 0.788） and （0.62 ± 0.06） g and （0.52 ± 0.02） g, respectively, at week 2 （P = 0.047）. At week 1, spermatids and sperm were more abundant in testes with pCAGGS-Epo than those in the control testes. At week 2, spermatids and sperm were hardly detected in either group. The G/S ratio was 23.27 ± 6.80 vs. 18.63 ± 5.30 at week 1 （P = 0.0078） and 7.16 ± 3.06 vs. 6.05 ± 1.58 at week 2 （P = 0.1471）, respectively. Conclusion： The transfer of Epo to rat testes by in vivo electroporation may reduce the risk of the germ cell loss caused by cryptorchidism.

Amelioration of carbon tetrachloride-induced cirrhosis and portal hypertension in rat using adenoviral gene transfer of Akt

AIM:To investigate whether a virus constitutively expressing active Akt is useful to prevent cirrhosis induced by carbon tetrachloride(CCl4).METHODS:Using cre-loxp technique,we created an Ad-myr-HA-Akt virus,in which Akt is labeled by a HA tag and its expression is driven by myr promoter.Further,through measuring enzyme levels and histological structure,we determined the efficacy of this Ad-myrHA-Akt virus in inhibiting the development of cirrhosis induced by CCl4in rats.Lastly,using western blotting,we examined the expression levels and/or phosphorylation status of Akt,apoptotic mediators,endothelial nitric oxide synthase(eNOS),and markers for hepatic stellate cells activation to understand the underlying mechanisms of protective role of this virus.RESULTS:The Ad-myr-HA-Akt virus was confirmed using polymerase chain reaction amplification of inserted Akt gene and sequencing for full length of inserted fragment,which was consistent with the sequence reported in the GenBank.The concentrations of Admyr-HA-Akt and adenoviral enhanced green fluorescent protein(Ad-EGFP)virus used in the current study were5.5×1011vp/mL.The portal vein diameter,peak velocity of blood flow,portal blood flow and congestion index were significantly increased in untreated,saline and Ad-EGFP cirrhosis groups when compared to normal control after the virus was introduced to animal through tail veil injection.In contrast,these parameters in the Akt cirrhosis group were comparable to normal control group.Compared to the normal control,the liver function(Alanine aminotransferase,Aspartate aminotransferase and Albumin)was significantly impaired in the untreated,saline and Ad-EGFP cirrhosis groups.The Akt cirrhosis group showed significant improvement of liver function when compared to the untreated,saline and Ad-EGFP cirrhosis groups.The Hyp level and portal vein pressure in Akt cirrhosis groups were also significantly lower than other cirrhosis groups.The results of HE and Van Gieson staining indicated that Akt group has better preservation of histological structure and less fibrosis than other cirrhosis groups.The percentage of apoptotic cell was greatly less in Akt cirrhosis group than in other cirrhosis groups.Akt group showed positive HA tag and an increased level of phosphorylated Akt as well as decreased levels of Fas.In contrast,Caspase-3 and Caspase-9 levels in Akt group were significantly lower than other cirrhosis groups.Noticeable decrease of DR5 andα-SMA and increase of phosphorylated eNOS were observed in the Akt group when compared to other cirrhosis groups.The NO level in liver was significantly higher in Akt group than other cirrhosis groups,which was consistent with the level of phosphorylated eNOS in these groups.CONCLUSION:This study suggest that Ad-myr-HA-Akt virus is a useful tool to prevent CCl4-induced cirrhosis in rat model and Akt pathway may be a therapeutic target for human cirrhosis.

Genome-wide analysis of the barley non-specific lipid transfer protein gene family

Non-specific lipid transfer proteins(nsLTPs) are small, basic proteins that are characterized by an eight-cysteine motif. The biological functions of these proteins have been reported to involve plant reproduction and biotic or abiotic stress response. With the completion of the barley genome sequence, a genome-wide analysis of nsLTPs in barley(Hordeum vulgare L.)(HvLTPs) will be helpful for understanding the function of nsLTPs in plants. We performed a genome-wide analysis of the nsLTP gene family in barley and identified 70 nsLTP genes,which can be divided into five types(1, 2, C, D, and G). Each type of nsLTPs shares similar exon and intron gene structures. Expression analysis showed that barley nsLTPs have diverse expression patterns, revealing their various roles. Our results shed light on the phylogenetic relationships and potential functions of barley nsLTPs and will be useful for future studies of barley development and molecular breeding.

Intrastriatal Gene Transfer of Vascular Endothelial Growth Factor Rescues Dopaminergic Neurons in a Rat Parkinson's Disease Model

To examine the ability of intrastriatal gene transfer of vascular endothelial growth factor 165 mediated by adenoviral vector to rescue dopaminergic neurons in a rat model of Parkinson＇s disease （PD）, we constructed recombinant replication-deficent adenoviral vectors carrying the gene of VEGF165 （Ad-VEGF）, and injected Ad-VEGF （or Ad-LacZ and PBS as controls） into the striatum of rats 7 days after the lesion by 6-hydroxydopamine. The rat rotational behavior analysis and tyrosine hydroxylase （TH） immunohistochemistry were performed to assess the change of dopaminergic neurons. Our results showed that the rats receiving Ad-VEGF injection displayed a significant improvement in apomorphine-induced rotational behavior and a significant preservation of TH-positive neurons and fibers compared with control animals, It is concluded that intrastriatal gene transfer by Ad-VEGF may rescue the dopaminergic neurons from degeneration in a rat model of PD.

Molecular Tissue Engineering: Applications for Modulation of Mesenchymal Stem Cells Proliferation by Transforming Growth Factor β_1 Gene Transfer

The effect of transforming growth factor β 1 (TGF β 1 ) gene transfection on the proliferation of bone marrow derived mesenchymal stem cells (MSC S ) and the mechanism was investigated to provide basis for accelerating articular cartilage repairing using molecular tissue engineering technology. TGF β 1 gene at different doses was transduced into the rat bone marrow derived MSCs to examine the effects of TGF β 1 gene transfection on MSCs DNA synthesis, cell cycle kinetics and the expression of proliferating cell nuclear antigen (PCNA). The results showed that 3 μl lipofectamine mediated 1 μg TGF β 1 gene transfection could effectively promote the proliferation of MSCs best; Under this condition (DNA/Lipofectamine=1μg/3μl), flow cytometry and immunohistochemical analyses revealed a significant increase in the 3 H incorporation, DNA content in S phase and the expression of PCNA. Transfection of gene encoding TGF β 1 could induce the cells at G0/G1 phase to S1 phase, modulate the replication of DNA through the enhancement of the PCNA expression, increase the content of DNA at S1 phase and promote the proliferation of MSCs. This new molecular tissue engineering approach could be of potential benefit to enhance the repair of damaged articular cartilage, especially those caused by degenerative joint diseases.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

AIM. To investigate the influence of heme oxygenase-1 （HO-1） gene transfer on the viability and function of cultured rat islets in vitro. METHODS： Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene （Ad- HO-1） or enhanced green fluorescent protein gene （Ad- EGFP）, and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/ propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index （SI） was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. RESULTS： After seven days culture, the viability of cultured rat islets decreased significantly （92% ± 6% vs 52% ± 13%, P 〈 0.05）, and glucose-stimulated insulin release also decreased significantly （6.47 ± 0.55 mIU/ L/30IEO vs 4.57 ± 0.40 mIU/L/3OIEO., 14.93 ± 1.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. Transfection of rat islets with adenoviral vectors at an 1±10 of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets（71% ± 15% vs 52% ± 13%, P 〈 0.05）. There was no significant difference in insulin release upon low glucose stimulation （2.8 mmol/L） among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group （P 〉 0.05）, while when stimulated by high glucose （16.7 mmol/L） solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively （12.50 ±2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ, 12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ, P 〈 0.05）. The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively （2.21 ± 0.02 vs 2.08 ± 0.05; 2.21 ± 0.02 vs 2.11 ± 0.03, P 〈 0.05）. CONCLUSION： The viability and function of rat islets decrease over time in in vitro culture, and heine oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.