天津医药

• 细胞与分子生物学 • 上一篇    下一篇

胱硫醚β-合酶/胱硫醚γ-裂解酶-硫化氢体系小干扰RNA对大鼠正常肝细胞系BRL凋亡的影响

闫骏1,李彦敏2,郭雪西1,章明放1   

  1. 1. 天津市第一中心医院
    2.
  • 收稿日期:2014-07-07 修回日期:2014-07-18 出版日期:2014-09-15 发布日期:2014-09-15
  • 通讯作者: 闫骏

  • Received:2014-07-07 Revised:2014-07-18 Published:2014-09-15 Online:2014-09-15
  • Contact: YAN Jun

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摘要： 目的 探讨内源性胱硫醚 β-合酶（CBS）/胱硫醚 γ-裂解酶（CSE）-硫化氢（H2S）体系在生理状态肝细胞凋亡中的影响。方法 培养大鼠正常肝细胞株BRL。小干扰RNA（siRNA）序列筛选：随机分组为阴性siRNA序列转染组、CBS siRNA序列转染组和CSE siRNA序列转染组，转染时间为48h或24h；siRNA转染后细胞凋亡检测：随机分组为siRNA阴性对照组、溶剂对照组、CBS siRNA组、CSE siRNA组、（CBS+ CSE）siRNA组，作用时间为转染后0、4、8、12、24 h；凋亡机制探讨：随机分组为siRNA阴性对照组、CBS siRNA组、CSE siRNA组、（CBS+ CSE）siRNA组，作用时间为转染后4、8、12、24 h；每组均为4个平行样。RT-PCR、Western blot检测BRL细胞中CBS、CSE的表达，siRNA基因静默CBS、CSE，去蛋白法检测H2S生成，Hoechst荧光染色、流式细胞术检测BRL细胞凋亡，荧光染色检测线粒体膜电位（MMP）变化，Western blot检测胞浆内细胞色素C（Cyt C）和激活型caspase 3蛋白表达变化。结果 BRL细胞株存在CBS、CSE表达。CBS/CSE siRNA转染后，细胞内源性H2S生成降低，细胞凋亡且凋亡率随时间延长而逐渐增加。结论 抑制内源性H2S体系可引起生理状态肝细胞凋亡，机制可能部分涉及凋亡线粒体途径。

关键词: 胱硫醚 β-合酶, 胱硫醚 γ-裂解酶, 硫化氢, 小干扰RNA, 凋亡, BRL细胞

Abstract: Objective To explore whether the inhibition of endogenous hydrogen sulfide, a novel and important gaseous transmitter generated in mammalian tissues mainly by Cystathionine β-synthase(CBS) or Cystathionine γ-lyase (CSE), induces apoptosis of the rat hepatic BRL cell line in physiological condition. Methods BRL cells were cultured, and divided randomly into several groups in different phases of the experiment. For example, negative-siRNA group, CBS siRNA groups and CSE siRNA groups were used to select the most efficient sequences of siRNAs at 48 or 24 hrs transfection. Solution group and (CBS+CSE) siRNA group were added to detect the variation of apotosis. The BRL cells line were observed and evaluated at 0, 4, 8, 12, and 24 hrs after siRNA transfection. When the mechanisms of apoptosis were detected, CBS/CSE siRNAs were transfected individually or jointly into BRL cells to examine the variation. The genic and protein expression of CBS/CSE were detected by RT-PCR and western blot. After transfection of CBS/CSE siRNA, BRL cells were detected apoptosis by Hoechst stain and Flow cytometry(FCM). The mitochondrial membrane potential(MMP) variation were observed by fluorescent staining. Western blot were used to examine the protein expression of cytochrome C and cleaved-caspase 3. Results CBS and CSE were observed in BRL cells. After transfection of CBS/CSE siRNA, endogenous H2S generation decreased and the apoptosis of BRL cells increased. Accordingly, the expression of intracytoplasm-Cyt C and cleaved-caspase 3 increased. Conclusion These results indicate that inhibition of endogenous H2S synthesis induces apoptosis of BRL cell under physiological condition. All these may involve in mitochondrial pathway of apoptosis.

Key words: Cystathionine β-synthase, Cystathionine γ-lyase, Hydrogen sulfide, siRNA, Apoptosis, BRL cells