Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Effect of human scavenger receptor SR-A I overexpression on uptaking ox-LDL and adhesion in 293T cells

AIM: To construct the recombinant eukaryotic expression plasmid pEGFP-C1-SR-A I for the high expression in 293T cells in order to identify functions of savenger receptor-A I (SR-A). METHODS: The primer was designed according to MSR1 cDNA and pEGFP-C1-SR-A I was constructed by standard molecular cloning technique and enzyme digestion. After sequencing, the plasmid was transfected into 293T cells by lipidosome method. The expression of scavenger receptor-A I was identified by RT-PCR and Western blotting. The foam cells were evaluated by the formation of lipid granules in the cells with oil red staining. Cell adhesion was analyzed by cell adhesion assay. RESULTS: 24 h after transfection, SR-A I mRNA was highly expressed and the high level of the protein was detected. The ratio of foam cell formation was doubled, the efficacy of cell adhesion was enhanced two times compared to the control group and the empty vector group. CONCLUSION: The recombinant eukaryotic expression plasmid has been constructed successfully with enhancing the function of uptake ox-LDL and adhesion in 293T cells by overexpression of SR-A I.     

Construction of eukaryotic expression vector and expression of outer membrane lipoprotein LipL41 gene of Leptospiria lai

AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell. METHODS:LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain, and was subcloned into vector pGEX-4T-1. After sequencing, LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion, it was transfected into COS7 cells by liposome. Its expression was analyzed by RT-PCR. RESULTS:A fragment of 1 011 bp was amplified, and sequence analysis showed it had a 98% homology with Leptospira kirschneri. The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed. A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR, but the cell transfected with blank plasmid did not show this band. CONCLUSIONS:The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA.     

Construction of eukaryotic expression vector of fusion gene CD80-IgG and its expression in Chinese hamster ovary cells

AIM: To construct the eukaryotic expression vector CD80-IgG by fusing the cDNA encoding extracellular portion of murine CD80 to the 5'-terminus of cDNA encoding Fc fragment of murine immunoglobulin G1 and to express the fusion protein in Chinese hamster ovary (CHO) cells. METHODS: The two cDNAs was amplified by PCR respectively from plasmid pcDNA/B7 containing the full-length cDNA of murine CD80 from murine spleen cells, and cloned to the eukaryotic expression vector pcDNA3.0 by directional cloning. The resultant recombinant plasmid pcDNA/CD80-IgG was transfected into CHO cells with liposome transfection reagent. The stably expressing cells were obtained by G418 screening. Western blot, Dot ELISA, and flow cytometry were used to detect the expression of the fusion protein and its immunological activity. RESULTS: DNA sequencing verified the correction of the construction of recombinant plasmid pcDNA/CD80-IgG. The expressed fusion protein was detected in the supernatant of transfected CHO cells and the molecular weight of the protein was similar to what we expected. Its immunological activity was also established. CONCLUSION: The recombinant plasmid pcDNA/CD80-IgG was successfully constructed and it expressed the fusion protein CD80-IgG.     

Knockdown of survivin expression using siRNA inhibits the growth of PC-3M cells

AIM: To study the inhibitory effects of survivin siRNAs on the growth of PC-3M cells. METHODS: Two pairs of DNA template coding siRNA against survivin were synthesized to construct two recombinant plasmids, pSi-sur1 and pSi-sur2. The two recombinants and the two controls, lipofectin and vacant plasmid were transfected into PC-3M cells. The expressions of survivin mRNA and protein were detected respectively by RT-PCR and Western blotting. Proliferation abilities were measured by MTT, and the cell cycle and apoptosis were assayed by FCM. RESULTS: After 72 h of transfection, the level of cell survivin mRNA in the two siRNA groups was 48%±6% (n=3) and 30%±5% (n=3) of that in lipofectin group, and expression of survivin protein were 38%±4% (n=3) and 36%±4% (n=3) respectively of that in lipofectin control. The proliferation rate of cells in pSi-sur1 and pSi-sur2 groups was also inhibited according to MTT, about 44.20%±2.08% (n=3) and 39.20%±1.93% (n=3) of that in lipofectin group. Cell numbers of G1 phase in two siRNA groups were significantly higher than that in two controls, while cells of G2 phase and S phase were much lower. Cell apoptosis was found in both siRNA groups. CONCLUSION: The two survivin siRNA significantly inhibit the expression of survivin in mRNA and protein levels, arrest the cell cycle in G1 phase, and suppress the growth of PC-3M cells and induce apoptosis in vitro.     

Molecular cloning of ING4 gene and ING4-induced apoptosis in HeLa cells

AIM: To isolate a gene encoding mouse ING4, construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and investigate its effects on HeLa cells in vitro. METHODS: The mouse ING4cDNA was amplified by RT-PCR from mouse liver. The eukaryotic expression vector pcDNA3.0-ING4 was constructed by DNA recombination technique. The recombinant plasmid pcDNA3.0-ING4 was identified by PCR, restriction enzyme digestion and DNA sequence analysis, then was transfected into HeLa cells by lipofectamine. The expression was determined by RT-PCR. Apoptosis was detected by fluorescence microscope with Hoechst33258 staining and laser scanning confocal microscope. Cell cycle distribution was measured with flow cytometry. RESULTS: RT-PCR product was about 750 bp specific fragment. Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed that results were about 750 bp, DNA sequencing revealed that ING4 cloning were successful. With Hoechst fluorescence staining, we found that the percentage of apoptotic rate in HeLa cells transfected with pcDNA3.0- ING4 (21.25%) was higher than that in HeLa cells transfected with pcDNA3.0 (8.91%,P<0.01). Apoptosis was also detected by laser scanning confocal microscope. Cell cycle analysis reavealed the cell number in S phase of HeLa cells transfected with pcDNA3.0- ING4 increased. CONCLUSION: The gene encoding mouse ING4 and construction of pcDNA3.0- ING4 eukaryotic expression vector were successfully obtained, ING4 could enhance apoptosis in HeLa cells.     

The antisense block of CROC-1 gene expression makes cells grow slower

AIM:To establish FL-CROC- 1 ^- cell line in which CROC- 1 gene expression was blocked and study the role of CROC- 1 gene in cell growth. METHODS:The appropriate length cDNA fragment of the recently identified human gene CROC- 1 which encodes ubiquitin-conjugating enzyme like protein(Ubc-like protein) was cloned into the reconstructed eukaryotic expression vector pMAMneo-amp^- by antisense strategy. The recombinant plasmid which can express CROC- 1 antisense RNA was selected by restriction enzyme map analysis. The antisense expression recombinant plasmid pMAM-antiCROC- 1 was then transfected into human amnion FL cells by a modified calcium phosphate-mediated transfection procedure and selected with MEM medium containing 400 μg/mL geneticin. Finally ,the growth rate of the G418 resistant FL-CROC- 1 ^- cell line was determined.RESULTS:When the antisense inhibition of CROC-1 gene expression was induced by dexamethasone,the growth rate of the FL-CROC-1^- cel line was obviously slower as compared with that of the control FL cell and FL-MAMneo cell which was established by transfection of plasmid pMAMneo-amp^-(P<0.05).CONCLUSIONS:FL-CROC- 1 ^- cell line was successfully established in this study and the result of FL-CROC- 1 ^- cell growth suppression suggests that CROC- 1 gene encoded Ubc-like protein plays a positive regulation role in cell growth.     

Construction of pNTAP-PRAK eukaryotic expression plasmid and its expression in HEK293 cells

AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.     

Subcellular localization of ZNF580-EGFP fusion protein

AIM: To construct a eukaryotic expression plasmid for enhanced green fluorescent protein (EGFP) and ZNF580 fusion protein, and study its subcellular localization in the transfected MGC803 cells. METHODS: The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame (1-172aa), ZNF580 amino terminus (1-93aa) and ZNF580 carboxyl terminus (94-172aa). The three cDNA segments of PCR were cloned into pGEM-T vector. Then they were subcloned respectively into plasmid pEGFP-C1 (enhanced green fluorescent protein). The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy. RESULTS: Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580 (1-172), pEGFP-ZNF580 (1-93) and pEGFP-ZNF580 (94-172) transgenic plasmid were correct. The fusion proteins of EGFP-ZNF580 (1-172) and pEGFP-ZNF580 (94-172) were localized in the nuclei. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed. The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus (C2H2 zinc finger domain).     

Construction and identification of eukaryotic dicistronic vector expressing TCA-12-2/TCB-7.1

AIM: To construct the recombinant dicistronic eukaryotic expression vector pDC315-TCA-12-2-TCB-7.1, which containing T cell antigen receptor (TCR) genes TCA-12-2 and TCB-7.1, and to transfer this recombinant vector into 293 cells to investigate the expression of TCA-12-2 and TCB-7.1. METHODS: The TCA-12-2 was obtained by RT-PCR from the T cells and the TCB-7.1 was amplified by PCR from plamid pcDNA3.1-TCB-7.1 that we constructed before. TCA-12-2 and TCB-7.1 was cloned into vector pIRES2-AcGFP1 firstly, then subcloned into vector pDC315. The recombinant plasmid pDC315-TCA-12-2-TCB-7.1 was verified by restriction enzyme digestion and sequencing, the positive recombinant plasmid was transferred into 293 cells using Lipofectamine 2000. The expressions of gene TCA-12-2 and TCB-7.1 were identified by RT-PCR and flow cytometry. RESULTS: Both TCA-12-2 and TCB-7.1 genes were constructed into eukaryotic expression vector pDC315 and the expressions of genes in 293 cells were detected successfully with RT-PCR and flow cytometry. CONCLUSION: The dicistronic expression vector pDC315-TCA-12-2-TCB-7.1 is successfully constructed and expressed.     

Cloning and expression of NK4 gene in Raji cells

AIM: To clone NK4 gene and to construct recombinant eukaryotic expression vector for observing its expression in transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue. NK4 gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct the recombinant eukaryotic expression vector pVITRO2-mcs-NK4. Raji cells were transfected by recombinant vector pVITRO2-mcs-NK4 and screened by homomycin B. The stable strain of NK4 gene expression was screened by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry and semisolid culture. RESULTS: The specific DNA fragment was detected by RT-PCR in Raji cells transfected with NK4 gene. The transfected Raji cells expressed NK4 mRNA and protein stably, which inhibited Raji cell proliferation, metastasis and invasion. CONCLUSION: NK4 gene is cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-NK4 successfully. NK4 gene in Raji cells expresses stably.     

Transition of WT1 isoforms inhibits proliferation and promotes apoptosis of human leukemia cell line HL-60

AIM: To study the potential effects of exogenous Wilms tumor 1 (WT1) isoforms on the proliferation and apoptosis of human leukemia cell line HL-60. METHODS: WT1 (17AA－/KTS－) gene obtained by RT-PCR was cloned into a PCDH1-MCS1-EF1-copGFP plasmid. The recombinant plasmid was confirmed by enzyme digestion and sequencing, and was transfected into HL-60 cells by LipofectamineTM 2000. The stable transformants were selected by G418 screening. WT1(17AA－/KTS－) expression was identified by real-time fluorescence quantitative RT-PCR and Western blotting. The proliferation of the cells was measured by MTT assay. The apoptosis of the cells was determined by morphological observation and flow cytometry analysis. RESULTS: The eukaryotic expression vector PCDH1-MCS1-EF1-copGFP-WT1 (17AA－/KTS－) was successfully constructed. The recombinant cells exhibited high mRNA and protein levels of WT1(17AA－/KTS－). The growth of recombinant cells was slower than that of HL-60 cells transfected with control vector and normal HL-60 cells. After exposed to As2O3 at 2 μmol/L for 48 h, both recombinant cells and control cells exhibited the morphological characteristics of apoptosis, but the former was more typical than the latter. The apoptosis was enhanced in the recombinant cells after the cells were exposed to As2O3 for 24 h. CONCLUSION: Exogenous WT1(17AA-/KTS-) isoform inhibits the proliferation and promotes the apoptosis of leukemic cells.     

Effect of sorcin expression on sensitivity of human glioma cells to cisplatin

AIM：To investigate the effect of sorcin expression on the sensitivity of human glioma cells to cisplatin. METHODS：pSilencerTM 3.1-H1-sorcin siRNA recombinant plasmid was constructed, and transfected into human glioma U251 cells. RT-PCR and Western blotting were used to analyze the expression of sorcin at mRNA and protein levels after transfection. The viability of U251 cells was measured by MTT assay. The protein expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) in U251 cells was detected by Western blotting. RESULTS：The plasmid pSilencerTM 3.1-H1-sorcin siRNA was successfully constructed, and was confirmed by restriction enzyme digestion and sequence analysis. The expression of sorcin at mRNA and protein levels was significantly decreased after sorcin siRNA was transfected into U251 cells (P<0.05). Inhibition of sorcin expression significantly decreased the viability of U251 cells treated with cisplatin (P<0.05), and the expression of P-gp and MRP1 proteins was also inhibited (P<0.05). CONCLUSION：Inhibition of sorcin expression increases the sensitivity of U251 cells to cisplatin by decreasing the expression of resistance-related proteins P-gp and MRP1, suggesting that sorcin may be associated with the resistance of glioma cells to cisplatin.     

Effects of TSG101 siRNA on the growth and drug sensitivity of SH-SY5Y cells

AIM:To investigate the effects of TSG101 siRNA on the growth and drug sensitivity of human neuroblastoma cell line SH-SY5Y.METHODS:The small interfering RNA eukaryotic expression vector specific to human TSG101 gene was constructed by gene recombination,then transfected into SH-SY5Y cells.Stable transfectants were obtained by G418 screening and further identified by RT-PCR and Western blotting analysis.The growth curve was made using MTT assay.Cell cycle distribution of the transfected cells was studied by flow cytometry and the proliferative indexes were calculated.The apoptosis after CDDP treatment was detected by DNA ladder and Annexin V/propidium iodide binding analyses.The expression of Bcl-2,Bax,P-gp and MRP were analyzed by Western blotting.RESULTS:mU6pro-TSG101 siRNA was successfully constructed and transfected into SH-SY5Y cells.As detected by MTT and flow cytometry,down-regulation of TSG101 significantly suppressed the proliferation of SH-SY5Y cells with a G1 cell cycle arrest,compared with that in control (P<0.05).As detected by DNA ladder and Annexin V/propidium iodide binding analyses,down-regulation of TSG101 significantly enhanced the sensitivity of SH-SY5Y cells to CDDP-induced apoptosis,compared with that in control (P<0.05).The expression of P-gp and Bcl-2 in transfected cells were decreased as compared with that in the control,while MRP and Bax were not.CONCLUSIONS:Down-regulation of TSG101 suppresses the proliferation of SH-SY5Y cells,and enhances the sensitivity of SH-SY5Y cells to conventional chemotherapeutic agents to a degree,suggesting TSG101 may be useful for gene therapy in the future.     

Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ eukaryotic expression vector and expression in NIH 3T3 cells

AIM: To construct a recombinant eukaryotic expression vector pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ and detect its expression in NIH 3T3 cells.METHODS: CD28-ζ cDNA was amplified from the plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/IgGFc/CD80 gene.The recombinant plasmids were transfected into NIH 3T3 cells,and resistant clones were obtained by G418 selection.The gene expression of the fusion protein was determined by RT-PCR and FACS.RESULTS: The recombinant eukaryotic vector was constructed successfully,determined by PCR and enzyme digestion analysis.The target gene was amplified from NIH 3T3 cells transfected with the vectors by RT-PCR.The FACS showed that recombinant protein was expressed in NIH 3T3 cells.CONCLUSION: Construction of pLNCX/anti-CD20scFv/IgGFc/CD80/CD28/ζ expression vector and its expression in NIH 3T3 cells lay the foundation for further research of generation of modified T lymphocytes to CD20 positive lymphoma.     

Effect of adipose differentiation-related protein on proliferation and apoptosis in H9c2 cells

AIM: To construct an adipose differentiation-related protein (ADRP) eukaryotic expression vector and to explore the effect of ADRP on apoptosis of H9c2 cells induced by palmitic acid (PA). METHODS: The ADRP gene obtained by the method of RT-PCR was cloned into pEGFP-C1 plasmid. The recombinant plasmid was transformed into E.coli DH5α for amplification. The recombinant plasmid was extracted from E.coli DH5α and transfected into H9c2 cells by Lipofectamine^TM2000. The stable transformants were selected by G418 screening. Expression of green fluorescent protein was observed under fluorescence microscope and the ADRP expression was identified by RT-qPCR and Western blotting analysis. The effect of PA on the proliferation of H9c2 cells was detected by MTT assay. The apoptotic percentage of H9c2 cells caused by PA was determined by flow cytometry. RESULTS: The eukaryotic expression vector pEGFP-C1-ADRP was successfully constructed. Green fluorescent was observed in the cells transfected with pEGFP-C1 or pEGFP-C1-ADRP under fluorescence microscope. RT-qPCR and Western blotting analysis showed that recombinant cells exhibited high mRNA and protein levels of ADRP. After treated with PA at different concentrations, the apoptosis rates and the proliferation inhibition of recombinant cells were both lower than those of the other two cells. CONCLUSION: The transfected H9c2 cells with stable ADRP expression were successfully established. The over-expression of ADRP prevents the cells from apoptosis and inhibition of proliferation caused by PA, indicating that ADRP plays a protective role in H9c2 cells.     

High level expression of recombinated hepatocyte growth factor in human umbilical cord mesenchymal stem cells

AIM: To construct a lentiviral vector encoding human hepatocyte growth factor (hHGF) for transfection,and to observe the expression of hHGF in human umbilical cord mesenchymal stem cells.METHODS: pUC-SRα/hHGF was subcloned into the expression vector pWPI to construct recombinant pWPI-hHGF.hHGF was identified by gene sequence.Recombinant lentivirus was produced by pWPI-hHGF,pAX2 and pMD2G altogether transient transfection into 293T cells using calcium phosphate method.The pWPI-hHGF and the contructed pWPI-GFP were transfected into human umbilical cord mesenchymal stem cells by the Lipofectamin 2000.Through counting by the fluorescent microscope,the efficiency of the transfection was identified.The expressions of hHGF and GFP in human umbilical cord mesenchymal stem cells were also detected.The concentration of hHGF in cell culture medium was determined by enzyme linked immunosorbent assay (ELISA).RESULTS: DNA sequence showed that hHGF cDNA was correctly inserted into pWPI vector.The positive rate of hHGF transfecting 293T cells was 100 %.Bright green fluorescence in the transfected cells was observed under the fluorescent microscope after 24 h transfection with lentiviral plasmid pWPI-hHGF-GFP,and the transfection rate reached 80%.The difference was distinct between the pWPI-hHGF group and control group in the secretive level of hHGF by Western blotting and the ELISA (P<0.01).CONCLUSION: The recombinant pWPI-hHGF plasmid was successfully constructed and efficient,stable and ectopic expression of hHGF was accomplished in human umbilical cord mesenchymal stem cells.