本研究探讨Wnt3a基因修饰对小鼠骨髓间充质干细胞(MSC)抗阿糖胞苷损伤的作用.通过重组腺病毒系统感染小鼠MSC,建立能够稳定高效表达Wnt3a基因的基因修饰MSC;在体外培养体系中加入不同浓度的阿糖胞分别诱导对基因修饰MSC与未经基因修饰MSC的损伤,设置对应的对照,通过CCK-8法、流式细胞术检测MSC的生长增殖以及凋亡情况;用Western blot测定MSC中与细胞凋亡有关的BCL-2蛋白的表达水平.结果表明,阿糖胞苷对基因修饰MSC的增殖抑制程度较未经基因修饰MSC明显减低,差异有统计学意义(p<0.05);去除阿糖胞苷后,基因修饰MSC的增殖生长能力在72小时后就开始恢复,而未经基因修饰MSC的增殖生长能力在72小时后仍然被抑制.在凋亡方面,阿糖胞苷诱导的基因修饰MSC的凋亡率明显降低(p<0.05),与未经基因修饰MSC相比,基因修饰MSC中BCL-2蛋白的表达上调(p<0.05).结论:Wnt3a基因修饰能明显减轻阿糖胞苷对小鼠骨髓MSC的损伤作用.%This study was aimed to investigate the protective effect of Wit3a gene modification on mouse bone marrow mesenchymal stem cells aganist the injury induced by Ara-C. The gene-modified MSC steadily expressing Wnt3a were established by adenovirus system. The acute direct damage effects of different concentrations of Ara-C on the unmodified MSC and the gene-modified MSC were assessed by using an in vitro culture system, and the corresponding controls were set. The proliferation and apoptosis of MSC exposed to Ara-C were detected by cell count kit-8 ( CCK-8 ) and flow cytometry. The expression of BCL-2 protein related with cell apoptosis was assayed by Western blot. The results indicated that as compared with unmodified MSC, Ara-C exhibited a less inhibitory effect on the proliferation of gene-modified MSC. There was obvious difference between unmodified MSC and gene-modified MSC (p ＜0.05). The proliferation of gene-modified MSC began to recover at 72 hours after removal of Ara-C. However, unmodified MSC showed sustained suppression of proliferation after withdrawal of Ara-C. In apoptosis, the apoptosis rate of genemodified MSC induced by Ara-C was significantly lower than those of unmodified MSC (p ＜ 0.05 ). In addition, the expression levels of BCL-2 protein in gene-modified MSC were up-regulated compared with unmodified MSC (p ＜ 0.05 ). It is concluded that Wnt3a gene modification can significantly mitigate the damage of mouse bone marrow MSC induced by Ara-C.
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