tMfn基因2对大鼠血管平滑肌细胞凋亡的影响

目的:采用腺病毒介导的去除穿膜区序列的大鼠线粒体融合素基因2(tMfn2)感染血管平滑肌细胞(VSMCs),研究其促VSMCs凋亡的作用及其相关的信号通路。方法:用携带大鼠线粒体融合素基因2(Mfn2)的腺病毒(Adv-Mfn2-GFP)和携带tMfn2基因的腺病毒(Adv-tMfn2-GFP)感染VSMCs,采用激光共聚焦显微镜技术观察tMfn2和Mfn2的融合蛋白在细胞中的定位;细胞凋亡ELISA方法、TUNEL染色法研究Mfn2、tMfn2对VSMCs凋亡的影响;Western Blot方法研究tMfn2促进VSMCs凋亡的相关信号通路。结果:激光共聚焦显微镜成像技术证实tMfn2融合蛋白主要游离于细胞质内,而Mfn2融合蛋白主要位于线粒体外膜。采用ELISA方法证实Adv-tMfn2-GFP感染组促进VSMCs凋亡作用比Adv-Mfn2-GFP感染组更显著;Adv-tMfn2-GFP感染组TUNEL染色的凋亡细胞较Adv-Mfn2-GFP感染组明显增多(P<0.05)。Western Blot结果显示,与空白对照组相比,Adv-Mfn2-GFP感染组、Adv-tMfn2-GFP感染组对应的P-Akt条带明显减弱,且后者更显著(P<0.05)。结论:tMfn2由于去除了穿膜区序列,其表达产物主要游离于细胞质中,更易于通过Ras-PI3K/Akt信号通路,抑制Akt的磷酸化,促进VSMCs凋亡,且作用较Mfn2更明显。

Effect of tMfn2 on the apoptosis of vascular smooth muscle cells

Objective:To investigate the effect of tMfn2 mediated by adenovirus on apoptosis of vascular smooth muscle cells (VSMCs) in rats.Method:VSMCs of rat were infected by adenovirus-mediated tMfn2(Adv-tMfn2-GFP) or adenovirus-mediated Mfn2(Adv-Mfn2-GFP). The location of fusion proteins of tMfn2 and Mfn2 in cell were observed by laser confocal microscopy. Cell death ELISA and TUNEL staining are used to investigate the effect on apoptosis of VSMCs. The corresponding signaling pathway of tMfn2 on promoting apoptosis of VSMCs is investigated with Westen blot.Result:Laser confocal microscopy showed that the fusion protein of tMfn2 was in intracytoplasm while that of Mfn2 in outer mitochondrial membrane. ELISA assay indicated that Adv-tMfn2-GFP was more remarkable on promoting apoptosis of VSMCs than Adv-Mfn2-GFP. TUNEL staining showed that there were more positive-apoptotic VSMCs in Adv-tMfn2-GFP than that in Adv-Mfn2-GFP (P<0.05). The result of Westen blot indicated that tMfn2 inhibited phosphorylation of Akt more effectively compared with Mfn2. Conclusion:tMfn2 can promote the apoptosis of VSMCs via the PI3K-Akt signaling pathway more effectively compared with Mfn2.