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《Chinese Journal of Neuromedicine》 2007-11
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Experimental study on transfection of adenovirus vector encoding green fluorescent protein into human bone marrow-derived mesenchymal stem cells

XU Gang XU Ru-xiang JIANG Xiao-dan ZHOU De-xiang YAO Peng-fei QIN Kun CAI Ying-qian ZOU Yu-xi QIN Ling-sha.Department of Neurosurgery;Neurosurgery Institute of Guangdong Province,Zhujiang Hospital,Southern Medical University,Guangzhou 510282,China  
Objective To explore the transfection of adenovirus encoding green fluorescent protein(Ad-GFP)vector into human bone marrow-derived mesenchymal stem cells(BMSCs),the dose-effect relationship between them and the impact on biological properties of BMSCs,and further investigate the feasibility of using Ad-GFP vector to construct gene-modifled BMSCs.Methods BMSCs were separated to be cultured in vitro.Flow cytometry was applied to detect the immune phenotype of cells;the adenovirus was obtained by packaging cell lines 293;BMSCs were transfected with Ad-GFP of various titers(1×10~3-1×10~(10)PFU/mL);cytometry was used to analyze the transfection efficiency;inverted microscope was applied to observe the morphological changes of cells;cell counting kit 8(CCK8)was applied to detect the activity of cell proliferation;β-mercaptoethanol was added after serum evacuation to induce directed differentiation of Ad-GFP-transfected BMSCs into neuron-like cells. Results CD34 and CD45,and CD29 and CD44,the surface markers of the third-to sixth-passage BMSCs were respectively negative and positive.When the virus titer was 1×10~7 PFU/mL,the transfection rate was 55% and when virus titers were 1×10~9 and 1×10~(10)PFU/mL,the transfection rate was 85%.In addition,pathological phenomena occurred to cells at the virus titer of 1×10~(10)PFU/mL.The fluorescent expression achieved its climax at 7 d and could still be seen at 28 d.BMSCs expressing Ad-GFP had three types of subpopulation structures under a fluorescence microscope;when the virus titer was≥1×10~6 PFU/mL,BMSCs proliferation was suppressed at the early stage,in a dose-dependent manner. BMSCs transfection with Ad-GFP could be differentiated into neuron-like cells with the induction ofβ-mercaptoethanol.Neuron-specific enolase(NSE)was positive.Conclusion Ad-GFP with suitable titers can infect BMSCs effectively,and has little impact on biological properties of cells and no impact on the differentiation-inducing function.BMSCs can serve as seed cells during the application of Ad-GFP vector system to the research of gene therapy.
【CateGory Index】: R329
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