A Study On The Effect Of BMSCs_-EGFP-tk As Mediator Of HSV1-tk/GCV Suicide Gene Therapy Directed Against A549in Vitro

OBJECTIVE:To construct containing HSV1-tk and EGFP gene Vector pDON-AI-2Neo-HSV1-tk-IRES2-EGFP and to observe the expression of HSV1-tk in mouse bone marrow mesenchymal stem cells. Then detecting the effect of BMSCs as mediator of HSV1-tk/GCV suicide gene therapy directed against A549cells in vitro.METHODS:The HSV1-tk cDNA fragments were obtained by polymerase chain re-action (PCR) from pHSV106and Bgl Ⅱ, Sal Ⅰ two restriction sites were added, aft-er T vector cloning,constructing the recombinant plasmid pHSV1-TK/18T; In the sa-me way, to construct the recombinant plasmid pDON-AI-2-Neo-HSV1-tk-IRES2-EG-FP. And the achievement of the package cell293T/tk with recombinant retroviral vector containing HSV1-tk gene.The recombinant plasmid was transfected into mouse bone marrow mesenchymal stem cells in vitro with lipofectamine transfection reagent and retroviral vector, then the expressions of EGFP in cells were observed by fluorescence microscopy and HSV1-tk gene was detected with RT-PCR. At last, the A549cells were co-cultured in direct contact with BMSCs-EGFP-tk, the effect was determined by MTT.RESULTS:①A length of1131bp with Bgl Ⅱ and Sal Ⅰ target gene sequences was obtained by PCR;②The packaged recombinant retroviral plasmids were detected by RT-PCR, the expression of HSV1-tk mRNA was determined;③In this two methods of lipofectamine transfection reagent and retroviral vector,the EGFP green fluorescent protein was detected after transfection, at the same time, the expression of HSV1-tk mRNA was determined.And effectively expressing HSV1-tk mRNA in mouse bone marrow mesenchymal stem cells in vitro.But the transfection efficiency of retroviral vector was obviously higher than the lipofectamine transfection reagent (P<0.05);④The biological characteristics of BMSCs-EGFP-tk were consistent with those of BMSCs and fluorescent light expression and HSVl-tk gene expression can persist at least15days;⑤MSCs、BMSCs-EGFP-tk、A549cells were cultured Separately,adding lug/mL GCV, the mortality is0.87.42%、0.48%; BMSCs-EGFP-tk: A549=2:1,adding lug/mL GCV, the theory mortality is58.44%, but actually,the mortality is90%. CONCLUSION:①The pHSVl-tk-IRES2-EGFP and pDON-AI-2-Neo-HSV1-tk-IRES2-EGFP expression plasmids were constructed successfully The package cells293T/tk with recombinant retroviral vector containing HSV1-tk gene were successfu-lly constructed), and transfected into mouse bone marrow mesenchymal stem cells in vitro successfully;②There is almost no difference between BMSCs-EGFP-tk and B-MSCs cells in biological characteristics;③The growth of A549cells have an obviou-sly inhibition and the bystander effect is outstanding in vitro after co-culture;④This experiment lays solid foundation for the future research.