Caspase-3 siRNA减少骨髓间充质干细胞稳定株细胞凋亡的实验研究

刘立宝; 黄蕾; 张军航; 廖洪映; 胡爱玲; 许世磊; 华平

doi:10.3969/j.issn.1674-7445.2014.05.005

Caspase-3 siRNA减少骨髓间充质干细胞稳定株细胞凋亡的实验研究

doi: 10.3969/j.issn.1674-7445.2014.05.005

1.
510630 广州, 中山大学附属第三医院心胸外科（刘立宝、黄蕾、张军航、廖洪映、胡爱玲、许世磊）
2.
中山大学孙逸仙纪念医院心脏外科（华平）

基金项目:

广东省科技计划项目基金 2007B050200011

详细信息

通讯作者:
华平,Email:huaping88@sina.com

中图分类号: R617
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- 被引次数: 0
出版历程
- 收稿日期: 2014-05-20
- 刊出日期: 2014-09-15

Experimental study on the Caspase-3 siRNA decreases apoptosis in stable cell lines of bone marrow mesenchymal stem cells

1.
Department of Cardio-Thoracic Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, China

摘要

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。
- Caspase-3 /
- 核糖核酸干扰 /
- 逆转录病毒 /
- 细胞凋亡 /
- 骨髓间充质干细胞
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.
- Caspase-3 /
- RNA interference /
- Retrovirus /
- Cell apoptosis /
- Bone marrow mesenchymal stem cell

HTML全文

图 1 MSC稳定株细胞和正常MSC的倒置荧光显微镜镜下的显像

注：A、B图为MSC-si-Caspase-3稳定株细胞（第3代），C、D图为正常MSC（第3代）

Figure 1. The stable cell lines of MSC and normal MSC's imaging under the inverted fluorescence microscope

下载: 全尺寸图片幻灯片

图 2 大鼠稳定株细胞与正常MSC中Caspase-3蛋白相对表达量比较

注：与正常MSC比较，^aPP<0.05

Figure 2. Comparison of Caspase-3-3 protein between the stable cell line of MSC and normal MSC

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图 3 大鼠稳定株细胞与正常MSC中Caspase-3 mRNA的比较

注：与正常MSC比较，^aPP<0.05

Figure 3. Comparison of Caspase-3 mRNA between the stable cell line of MSC and normal MSC

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图 4 稳定株细胞与正常MSC凋亡的比较

Figure 4. Comparison of apoptosis between the stable cell line of MSC and normal MSC

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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
Caspase-3	下游:AGGAGATGCCACCTCTCCTT	20	118
β-actin	上游:CCTAGGCACCAGGGTGTGAT	20	122
β-actin	下游:TTGGTGACAATGCCGTGTTC	20	122

下载: 导出CSV

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