中国组织工程研究2018，Vol.22Issue(17)：2650-2655,6.DOI:10.3969/j.issn.2095-4344.0515
腺病毒介导增强型绿色荧光蛋白基因转染兔骨髓间充质干细胞的效率及毒性
Transferring adenovirus vector-mediated enhanced green fluorescent protein gene into rabbit bone marrow mesenchymal stem cells: effectiveness and toxicity
摘要
Abstract
BACKGROUND: The recombinant adenovirus has certain toxic reactions to cell growth and survival, but the determination of the optimal multiplicity of infection (MOI) is not well documented. OBJECTIVE: To study the effectiveness and toxicity of transfection of bone marrow mesenchymal stem cells (BMSCs) by adenovirus vectors with different MOIs, and to explore the effect on osteogenic capability of the cells. METHODS: BMSCs at passage 5 were infected with enhanced green fluorescent protein (EGFP) via adenovirus vectors with different MOI (0, 50, 100, 150, 200 and 250). Cell morphology and mortality rate were observed and calculated using trypan blue staining method at 24 hours after cell transfection. The transfection efficiency and the relative mRNA expression of EGFP and Runx2 were analyzed respectively by inverted florescent microscopy and real-time quantitative PCR at 48 hours after cell transfection. The activity of BMSCs transfected with different MOIs was evaluated by MTT, and the optimal MOI was determined thereafter. RESULTS AND CONCLUSION: With the increase of MOI, BMSCs showed decreased adherent ability, and even some cells aged and died. The mortality rate of BMSCs transfected for 24 hours at a MOI of 0 to 250 was 5.80%, 6.67%, 7.95%, 7.76%, 10.35% and 11.18%, respectively, indicating that the mortality rate of BMSCs is positively correlated with the MOI. The transfection efficiency changed insignificantly when the MOI was greater than 100. On the contrary, the cellular viability and osteogenic differentiation capability of rabbit BMSCs were receded when the MOI level was up to 200-250. The study discovered that the suitable scope of MOI to transfect BMSCs is 50-150, and the optimal MOI is 100.关键词
干细胞/骨髓间充质干细胞/腺病毒/感染复数/细胞毒性/国家自然科学基金分类
医药卫生引用本文复制引用
武成聪,荣树,任静,吴铮,刘涛,刘克廷,朱博,黄合飞,王芳..腺病毒介导增强型绿色荧光蛋白基因转染兔骨髓间充质干细胞的效率及毒性[J].中国组织工程研究,2018,22(17):2650-2655,6.基金项目
国家自然科学基金资助项目(31160199) （31160199）
云南省教育厅科学研究基金项目(2016ZDX074) the National Natural Science Foundation of China, No. 31160199 （2016ZDX074）
the Science Research Foundation of the Department of Education of Yunnan Province, No. 2016ZDX074 （）
