摘要
背景:重组腺病毒对细胞生长、存活存在一定的毒性反应,其最适感染复数的确定相关研究较少。目的:研究重组腺病毒在不同感染复数下其转染效率、细胞毒性及对骨髓间充质干细胞成骨分化潜能的影响。方法:携带增强型绿色荧光蛋白基因的重组腺毒载体分别以感染复数为0,50,100,150,200,250转染第5代骨髓间充质干细胞。转染24 h后观察细胞形态,锥虫蓝染色计数死亡率;转染48 h后荧光倒置显微镜下计算转染率,q PCR检测EGFP及Runx2基因表达量。MTT法评价不同感染复数转染对细胞活性的影响,确定最佳感染复数。结果与结论:(1)随感染复数值增加,骨髓间充质干细胞贴壁能力减弱,部分细胞老化、死亡;(2)以感染复数值为0,50,100,150,200,250转染24 h,其细胞死亡率依次为5.80%,6.67%,7.95%,7.76%,10.35%,11.18%,死亡率与感染复数值成正相关;(3)当感染复数值为100时即可达到最大转染效率,但当感染复数增大至200时,细胞活性即受到影响;当感染复数达到250时,骨髓间充质干细胞成骨分化潜能受到影响;(4)通过上述结果可以认为,腺病毒转染兔骨髓间充质干细胞的感染复数安全范围为50-150,其最适感染复数为100。
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 m RNA 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.
作者
武成聪
荣树
任静
吴铮
刘涛
刘克廷
朱博
黄合飞
王芳
Wu Cheng-cong;Rong Shu;Ren Jing;Wu Zheng;Liu Tao;Liu Ke-ting;Zhu Bo;Huang He-fei;Wang Fang(First Department of Orthopaedics,Qujing No.1 Hospital,Qujing 655000,Yunnan Province,China;Department of Pathology,Qujing Second Hospital,Qujing 655000,Yunnan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2018年第17期2650-2655,共6页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金资助项目(31160199)
云南省教育厅科学研究基金项目(2016ZDX074)~~
