去除穿膜区序列的线粒体融合素2基因通过线粒体凋亡路径促进大鼠血管平滑肌细胞凋亡  

tMfn2 gene transfer promotes vascular smooth-muscle cells apoptosis via activation of the mitochondrial cell death pathway

作　　者：赵丽[1] 周炜[1] 王四坤[1] 廖华[1] 张文娟[1] 陈光慧 郭小梅[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院心血管内科,武汉430030 [2]美国国立卫生研究所

出　　处：《中华心血管病杂志》2009年第7期639-643,共5页Chinese Journal of Cardiology

基　　金：国家自然科学基金资助项目（30570730）

摘　　要：目的研究去除穿膜区序列的大鼠线粒体融合素2（tMfn2）基因对大鼠血管平滑肌细胞（VSMC）凋亡的影响及其相关的信号通路。方法用携带tMfn2基因和线粒体融合素2（Mfn2）基因的重组腺病毒（Adv—tMfn2和Adv—Mfn2）感染VSMC。采用流式细胞术、细胞凋亡ELISA、TUNEL染色等方法检测tMfn2对VSMC凋亡的影响。Western blot分析磷酸化蛋白激酶B（p-Akt）以及线粒体凋亡路径中B细胞淋巴瘤／白血病-2蛋白（Bcl-2）、Bcl-2相关的X蛋白（Bax）、有活性的天冬氨酸特异一半胱氨酸蛋白酶9（cleaved caspase-9）的表达变化。结果流式细胞仪检测和ELISA结果表明，tMfn2促VSMC凋亡的作用显著强于Mfn2，且呈时产依赖性[72h凋亡率分别为（79．2±0．2）％和（65．0±1．2）％，P〈0．01]。TUNEL染色发现tMfn2组的凋亡细胞明显多于Mfn2组（P〈0．01）。Western blot结果显示，tMfn2和Mfn2组中p-Akt水平均明显降低，但前者作用更显著（P〈0．01）。进一步检测线粒体凋亡路径中的相关蛋白，tMfn2组的Bax蛋白表达显著升高、Bcl-2蛋白表达显著降低，且cleaved caspase-9的活性明显增强，较Mfn2诱导凋亡的作用更强（P〈0．01）。结论与Mfn2相比，tMfn2促进VSMC凋亡的作用更强，其机制与抑制Akt磷酸化并激活线粒体凋亡途径有关。Objective To investigate the effect of tMfn2 gene transfer on promoting the apoptosis of vascular smooth-muscle cells （VSMCs）. Methods VSMCs were infected by adenovirus-mediated tMfn2 （Adv-tMfn2） or adenovirns-mediated Mfn2 （Adv-Mfn2）. FACS analysis, cell death ELISA and TUNEL staining were used to investigate the role of tMfn2 and Adv-Mfn2 gene transfer on VSMCs apoptosis. Western blot was used to analyze the protein expression of p-Akt, Bcl-2, Bax and cleaved capase-9. Results FACS and ELISA results showed that tMfn2 was superior to Mfn2 in promoting VSMCs apoptosis and tMfn2 gene transfer induced VSMCs apoptosis in a time-dependent manner（ P 〈 0. 01 ）. TUNEL staining evidenced that there were more positive-apoptotic VSMCs in tMfn2 group than that in Mfn2 group （P 〈0.01 ）. The protein expressions of phosphorylated Akt and Bcl-2 were significantly decreased, whereas Bax and cleaved caspased-9 protein expressions were significantly upregulated in tMfn2-transfected VSMCs. Conclusions tMfn2 transfer promoted apoptosis of VSMCs in a time dependent manner via the PI3K-Akt signaling pathway and mitochondrial apoptotic pathway.

关 键 词：肌 平滑 血管 细胞凋亡 腺病毒科 线粒体融合素2 

分 类 号：R686[医药卫生—骨科学]