Research Of Glioma Treatment By Using Bone Marrow Stromal Cells Transfected With Gene HSVtk

Objective: Glioblastoma is the most common malignancy in human brain,of which the treatment effect is unsatisfactory for the moment,no matter the surgical treatment or the radiotherapy and the chemotherapy.Therefore,the gene therapy is becoming more and more concerned.Tumor suicide gene therapy system of HSVtk/GCV(herpes simplex virus thymidine kinase/ganciclovir)plays a significant role in anti-tumor.However,due to the low transduction efficiency in vivo of HSVtk,the appropriate carrier cell has not been found by the introduction of HSVtk gene in vitro,which directly hinders the further clinical application.In recent years,research shows that bone marrow mesenchymal stem cells(BMSC)are capable of carrying and transporting therapeutic genes during the treatment.Besides,it has been found that the BMSC-tk,the carrier of HSVtk gene,has a favorable bystander effect with different kinds of glioma cells.This study is designed to further investigate the antineoplasmic effect of BMSC-tk.Methods: C6(rat glioma cells)culture.The extraction,morphological identification anculture of primary BMSC.The orienteering ability of BMSC to C6 was verified bTranswell migration test.Over vector p EX-4-herpesvirus were transfected into C6 anBMSC cell.The observation of transfection effect between those two kinds of cells waperformed.The susceptibility in vitro of C6-tk and BMSC-tk to GCV was tested bCCK-8(cell counting kit-8).C6 and BMSC-tk were cultured together in the nutrient fluiwhich had GCV of different concentration(0~100?g/ml).The antineoplasmic activitassessment was completed in the process of testing the number of surviving cells bCCK-8 analytical method.Results: Numbers of cell permeating septum in conditioned medium was more than control group(P<0.001).The transfection efficiency of TK plasmid was over 20% in C6 glioma cells,while it was under 10% in BMSCs,and there was less GFP expression in BMSC.There was a distinct decrease in both C6-tk and BMSC-tk cell viability with an increasing GCV concentration;Comparing with control group,C6-tk cell viability started to decrease when GCV concentration is above 0.1ug/ml,while it happened to BMSC-tk cells when GCV concentration is above 0.3ug/ml;At a 10ug/ml GCV concentration,both C6-tk and BMSC-tk cells reached the lowest viability(P<0.001)and dropped no longer even with a higher GCV concentration(P>0.05).With a GCV concentration of 1ug/ml,3ug/ml,10ug/ml,30ug/ml and 100ug/ml,there was a distinct decrease in BMSC-tk+C6 cell viability(P<0.05,P<0.01,P<0.001,P<0.001,P<0.001).When GCV was 10ug/ml,BMSC-tk+C6 cells reached the lowest viability and stopped dropping when GCV concentration was increased.When GCV was 10ug/ml,BMSC-tk+C6 cells had lower OD value than BMSC-NC+C6 cells in CCK8 and the difference was notably higher than that between BMSC-tk cells and BMSC-NC cells(P<0.001).Conclusion: 1.There was migratory activity towards C6 cells in BMSCs.2.In vitro,C6-tk cells were sensitive to GCV and was most significant when GCV was 10ug/ml.3.In vitro,BMSC-tk cells were sensitive to GCV and was most significant when GCV was 10ug/ml.4.In vitro,BMSC-tk cells shown killing effect to C6 cells,and the minimum GCV level of 10ug/ml can reach the best lethal efficiency.