腺病毒介导TWEAK对大鼠骨髓间充质干细胞影响的实验研究

Osteogenic Effect of TWEAK Gene on Rats Bone Marrow Mesenchymal Stem Cells Induced by Adenovirus

目的:探讨腺病毒介导的TWEAK基因对骨髓间充质干细胞(Bone M esenchym al Stem Cells,BMSCs)细胞的影响。方法:构建含有TWEAK基因的腺病毒载体Ad5CMV-TWEAK和含绿色荧光蛋白的对照腺病毒载体Ad5CMV-EGFP;利用全骨髓贴壁法分离纯BMSCs,通过转染Ad5CMV-EGFP确定病毒对BMSCs的转染效率;以未经转染的BMSCs细胞为空白对照组,以转染Ad5CMV-EGFP为实验对照组,以转染Ad5CMV-TWEAK为实验组,通过计数细胞和测定细胞碱性磷酸酶含量观察转染TWEAK对BMSCs增殖和骨形成能力的影响。结果:Ad5CMT-EGFP对大鼠BMSCs的感染率在300partic les/cell时可达到85%;感染后72小时组实验组与空白对照组差异有统计学分析意义(P<0.05),实验对照组与空白对照组差异无统计学分析意义;感染后15 d内各时间点3组间碱性磷酸酶的分泌量经t检验,差异无统计学意义。结论:腺病毒介导的TWEAK基因对于BMSCs具有增殖作用,但并不通过碱性磷酸酶影响BMSCs成骨能力,其在骨形成中的作用需要进一步探讨。

Objective： To study the osteogenic effect of TWEAK gene on the rat BMSCs mediated by adenovirus vector. Methods： Firstly, we cultured rats BMSCs, then BMSCs were infected with AdSCMV- EGFP containing enhanced green fluorescence protein gene （EGFP） , in order to obtain the definite titer of this adenovirus vector. The BMSCs were divided into three groups ： uninfected group as negative control, infected AdSCMV - EGFP group as experiment control, infected AdSCMV - TWEAK as experiment group. 5 × 10^3 cells/well were plated in 96 well plate. Cells were harvested and counted every day after infection. The average number of cells for each day after infection was calculated after each growth assay was completed by combining data from 3 different groups. The ALP activity and the ALP gene expression were detected with the ALP test kit and modified Kaplow staining test. Results： Proper titer was 300particles/cell, and rats BMSCs could be infected 85% in this titer. The cell growth assay showed that BMSCs amount increased after infected AdSCMV - TWEAK and there was obvious difference compared with control group, and the proliferation ratio was restrained after 6d. The volume of the ALP from ld to 15d; there was not significant difference between infection groups and control groups. Conclusion： TWEAK can promote BMSCs proliferation, but can not significantly influence the BMSCs osteogenic differentiation.