基础医学与临床 ›› 2016, Vol. 36 ›› Issue (10): 1323-1328.

• 研究论文 •    下一篇

siRNA沉默mfn2表达对睾酮抑制大鼠血管平滑肌源性泡沫细胞形成的影响

周炜,陈曼华   

  1. 武汉市中心医院
  • 收稿日期:2015-12-03 修回日期:2016-03-25 出版日期:2016-10-05 发布日期:2016-09-27
  • 通讯作者: 周炜 E-mail:wzhou@live.cn
  • 基金资助:
    国家自然科学基金;湖北省自然科学基金

Effects of mfn2 siRNA on vascular smooth muscle cells derived foam cells formation inhibited by testosterone

  • Received:2015-12-03 Revised:2016-03-25 Online:2016-10-05 Published:2016-09-27

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摘要： 目的 研究线粒体融合素2基因（mfn2）在睾酮抑制大鼠血管平滑肌源性泡沫细胞形成过程中的作用。方法 设计并化学合成靶向mfn2基因的小分子干扰RNA（siRNA），脂质体法将mfn2-siRNA转染大鼠血管平滑肌细胞（VSMCs），实时荧光定量PCR和免疫印迹法检测mfn2 mRNA及蛋白的表达。培养大鼠VSMCs，利用氧化型低密度脂蛋白（ox-LDL）诱导形成泡沫细胞。实验分组：1）对照组；2）睾酮组；3）睾酮+阴性对照siRNA组；4）睾酮+mfn2-siRNA组。后3组预先加入睾酮和（或）siRNA预处理12 h，再与ox-LDL共培养48 h。油红O染色及酶荧光法检测细胞内脂质含量，免疫印迹法检测细胞内Mfn2、清道夫受体CD36和ATP结合盒转运蛋白A1（ABCA1）的表达。 结果 转染mfn2-siRNA后12、24、36和48 h均能有效沉默mfn2 mRNA和蛋白的表达（P<0.05）。与对照组比较，睾酮组及睾酮+阴性对照siRNA组脂质蓄积减少，总胆固醇、游离胆固醇和胆固醇酯的含量均降低（P<0.05），Mfn2和ABCA1表达增加（P<0.05），CD36表达降低（P<0.05），细胞泡沫化受到抑制。与睾酮+阴性对照siRNA组比较，睾酮+mfn2-siRNA组脂质蓄积增加，总胆固醇、游离胆固醇和胆固醇酯的含量均增加（P<0.05），Mfn2和ABCA1表达降低（P<0.05），CD36表达增加（P<0.05）。 结论 睾酮通过调节mfn2及其下游CD36和ABCA1表达，抑制ox-LDL诱导下VSMCs转变为泡沫细胞。

关键词: 小分子干扰RNA, 线粒体融合素2, 睾酮, 血管平滑肌细胞, 泡沫细胞

Abstract: Objective To evaluate the effects of mitofusin 2(mfn2) gene silencing by specific small interference RNA(siRNA) on vascular smooth muscle cells(VSMCs) derived foam cells formation inhibited by testosterone. Methods The three double-strand siRNAs targeting mfn2 gene were designed and synthesized, then transfected into rat VSMCs by positive ion liposome Lipofectamine 2000. The mRNA and protein expression levels of mfn2 in VSMCs were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Then, VSMCs were divided into control group, testosterone group, testosterone+control-siRNA group, and testosterone+mfn2-siRNA group. Effects of testosterone with or without siRNA on the quantity of intracellular lipid of the VSMCs were detected with Oil red O staining and enzymatic fluorometric method. The expression of the Mfn2 was analyzed with Western blotting. We also examined the effect of testosterone with or without siRNA on the cholesterol transport related protein(CD36 and ABCA1). Results After mfn2-siRNAs were successfully transfected into rat VSMCs, compared to the non-targeting siRNA，the mfn2 mRNA and protein expression levels in VSMCs were significantly decreased at 12, 24, 36, 48 h(P<0.05). Compared with the control group, lipid deposition was reduced significantly in the testosterone with or without control-siRNA groups(both P<0.05). Testosterone markedly increased the expression of Mfn2 and ABCA1(both P<0.05), while suppressed the expression of CD36(P<0.05) in ox-LDL stimulated VSMCs. Moreover, such suppressive effects of testosterone on foam cells formation could partly been reversed by the mfn2-siRNA(P＜0.05), suggesting an involvement of mfn2 in the mechanism. Conclusions Testosterone inhibited the formation of VSMCs derived foam cells induced by ox-LDL partly via the Mfn2 dependent manner by down-regulation of CD36 and up-regulation of ABCA1 expression.

Key words: small interfering RNA, mitofusin 2, testosterone, vascular smooth muscle cells, foam cells

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