tMfn2基因诱导血管平滑肌细胞的凋亡(英文)

tMfn 2 gene induces apoptosis of vascular smooth muscle cells

背景:线粒体融合素2蛋白,主要通过磷脂酰肌醇3激酶/蛋白激酶B诱导血管平滑肌细胞凋亡。去除穿膜区序列的线粒体融合素2蛋白,相对分子质量减小41%,诱导凋亡作用可能更强。目的:观察比较去除穿膜区序列的大鼠线粒体融合素基因2对大鼠血管平滑肌细胞凋亡的影响及其相关的信号通路。设计、时间及地点:基因水平的对比观察实验。于2008-01/10在华中科技大学同济医学院附属同济医院分子心脏病中心实验室完成。材料:大鼠血管平滑肌细胞及携带半乳糖甘酶基因、携带线粒体融合素2基因和携带去除穿膜区序列线粒体融合素2基因的重组腺病毒均由陈光慧教授惠赠。方法:将大鼠血管平滑肌细胞传代培养3～10代后随机分为4组,①空白对照组:不加干预。②携带半乳糖甘酶基因的对照组:感染携带半乳糖甘酶基因的重组腺病毒。③携带线粒体融合素2基因的实验组:感染携带线粒体融合素2基因的重组腺病毒。④携带去除穿膜区序列线粒体融合素2基因的实验组:感染携带去除穿膜区序列线粒体融合素2基因的重组腺病毒。主要观察指标:①重组腺病毒感染血管平滑肌细胞24h后观察完整的和去除穿膜区序列的线粒体融合素2基因的表达情况。②感染后24,48和72h采用流式细胞术、酶联免疫吸附法观察细胞凋亡情况。③免疫印迹分析病毒感染血管平滑肌细胞24h后磷酸化蛋白激酶B表达变化。结果:①感染后完整的和去除穿膜区序列的线粒体融合素2基因在血管平滑肌细胞中均有表达。②去除穿膜区序列的线粒体融合素2基因诱导血管平滑肌细胞凋亡的作用显著增强,且呈时间依赖性(P<0.01)。③两个实验组中磷酸化蛋白激酶B水平均明显降低,但去除穿膜区序列的线粒体融合素2基因实验组更显著(P<0.01)。结论:去除穿膜区序列的线粒体融合素基因2诱导血管平滑肌细胞凋亡的作用更强,其机制与抑制蛋白激酶B磷酸化有关。

BACKGROUND： Mitofusin2 （Mfn2） profoundly inhibits proliferation and induces apoptosis of vascular smooth muscle cells （VSMCs） through phosphatidylinositol 3 kinase/protein kinase B. Notably, tMfn2, with the transmembrane domain deleted, has a 41%-reduced molecular weight, which possibly exhibits a stronger effect on inducing apoptosis. OBJECTIVE： To investigate the effect of rat tMfn2 gene, with the transmembrane domain deleted, on promoting the apoptosis of VSMCs, and to determine related signal pathway. DESIGN, SETTING AND TIME： A controlled observational study at a gene level was performed in the central laboratory, Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology between January and October in 2008. MATERIALS： Rat VSMCs and the recombinant adenovirus containing LacZ, Mfn2 or tMfn2 was offered by Professor Chen as a gift. METHODS： The rat VSMCs cultured at 3-10 passages were divided into 4 groups. ①Blank control group： No interventions.② VSMCs were infected by adenovirus-mediated LacZ, Mfn2, and tMfn2, respectively. MAIN OUTCOME MEASURES： ①Expressions of Mfn2 and tMfn2 following the VSMCs were infected with recombinant adenovirus for 24 hours. ②The apoptosis of VSMCs was determined with flow cytometry and enzyme linked immunosorbent assay at 24, 48 and 72 hours following infection. ③Western blot was used to analyze the expression of phosphorylated AKT following the VSMCs were infected with recombinant adenovirus for 24 hours. RESULTS： ①Both Mfn2 and tMfn2 were expressed in the VSMCs. ②The tMfn2 was superior to Mfn2 in promoting the apoptosis of VSMCs in a time-dependent manner （P 〈 0.01 ）. ③The protein expression of phosphorylated AKT remarkably decreased two groups, especially significant in tMfn2-infected group （P 〈 0.01 ）. CONCLUSION： The tMfn2 can induce the apoptosis of VSMCs more effectively via the inhibition of phosphorylated AKT signaling pathway.