Transferring adenovirus vector-mediated enhanced green fluorescent protein gene into rabbit bone marrow mesenchymal stem cells: effectiveness and toxicity
10.3969/j.issn.2095-4344.0515
- VernacularTitle: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
- Author:
Cheng-Cong WU
1
;
Shu RONG
;
Jing REN
;
Zheng WU
;
Tao LIU
;
Ke-Ting LIU
;
Bo ZHU
;
He-Fei HUANG
;
Fang WANG
Author Information
1. 曲靖市第一人民医院骨一科
- From:
Chinese Journal of Tissue Engineering Research
2018;22(17):2650-2655
- CountryChina
- Language:Chinese
-
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.
