Experimental study on the Caspase-3 siRNA decreases apoptosis in stable cell lines of bone marrow mesenchymal stem cells
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摘要:
目的 探讨在体外采用Caspase-3小干扰核糖核酸(siRNA)减少骨髓间充质干细胞(MSC)稳定株细胞凋亡的方法,以构建稳定表达的MSC细胞株。 方法 使用逆转录病毒包装系统,在293FT细胞中进行包装生产含Caspase-3 siRNA的病毒颗粒,然后转染给大鼠MSC,对转染后的细胞进行筛选并扩增培养得到稳定株细胞。根据逆转录病毒的特性,利用蛋白质印迹法(Western blot)和实时荧光定量逆转录聚合酶链反应(RT-QPCR)对细胞株进行稳定表达的鉴定,用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定(TUNEL)法对稳定株细胞和正常MSC进行凋亡检测并进行定位比较。 结果 与正常细胞比较,MSC稳定株细胞中的Caspase-3蛋白表达量和mRNA含量明显降低;在同样体外环境下,MSC稳定株细胞的凋亡数量比正常细胞明显减少。 结论 利用逆转录病毒包装系统可得到稳定表达Caspase-3 siRNA的MSC稳定株细胞。MSC稳定株细胞比正常细胞凋亡明显减少。 Abstract:Objective To explore a method of using Caspase-3 small interference ribonucleic acid (siRNA) in vitro to decrease the apoptosis in stable cell lines of bone marrow mesenchymal stem cell (MSC) in order to establish MSC cell lines with stable expression. Methods Virus particles containing Caspase-3 siRNA were generated in 293FT cells by retroviral packaging system, then were transfected into MSC of rats. And the transfected cells were screened and cultured to get the stable MSC lines. According to the characteristics of retrovirus, the stable expression of the cell lines was identified by Western blot and real-time fluorescent quantitative PCR (RT-QPCR). The apoptosis of stable cell lines and normal MSC were detected by terminal dexynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and their differences were compared. Results Compared with the normal cells, the caspase-3 protein expression and mRNA content of stable MSC lines were significantly reduced. In the same condition in vitro, the apoptosis quantity of stable MSC lines significantly decreased compared with the normal cells. Conclusion Stable cell lines of MSC with stable expression of Caspase-3 siRNA can be obtained by retroviral packaging system. The apoptosis quantity of stable MSC Lines significantly decreased compared with the normal cells. -
Key words:
- Caspase-3 /
- RNA interference /
- Retrovirus /
- Cell apoptosis /
- Bone marrow mesenchymal stem cell
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表 1 本实验所用基因的引物序列
Table 1. Primer sequences of genes in the experiment
基 因 引物序列(5’to3’) 引物长度(bp) 扩增长度(bp) Caspase-3 上游:CGATTATGCAGCAGCCTCAA 20 118 下游:AGGAGATGCCACCTCTCCTT 20 β-actin 上游:CCTAGGCACCAGGGTGTGAT 20 122 下游:TTGGTGACAATGCCGTGTTC 20 -
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