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背景:线粒体融合素2基因作用于血管平滑肌细胞Ras蛋白,通过胞外信号调节蛋白激酶1/2通路抑制细胞增殖。线粒体融合素2基因氨基酸序列第442位丝氨酸为蛋白激酶A磷酸化位点,与其磷酸化状态密切相关,可能参与其功能调控。目的:观察大鼠线粒体融合素2基因在去除蛋白激酶A磷酸化位点后对大鼠血管平滑肌细胞增殖的影响及其相关信号通路。方法:利用已构建的携带绿色荧光蛋白基因、线粒体融合素2基因和去除蛋白激酶A磷酸化位点的线粒体融合素2基因的3种重组腺病毒,感染大鼠主动脉血管平滑肌细胞,将其传代培养3～10代后以抽签法随机分为4组:①不加干预的对照组。②感染携带绿色荧光蛋白的对照组(Adv-GFP组)。③感染携带线粒体融合素2基因的实验组(Adv-Mfn2组)。④感染携带去除蛋白激酶A磷酸化位点的线粒体融合素2基因的实验组(Adv-Mfn2-PKA(△)组)。激光共聚焦显微镜观察完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在细胞中的定位。Westernblot检测p-ERK1/2表达水平及完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中的表达。MTT法绘制细胞生长曲线。结果与结论:完整的和去除蛋白激酶A磷酸化位点的线粒体融合素2基因在血管平滑肌细胞中均表达蛋白特异性条带。两种基因表达产物都主要分布于线粒体外膜。与对照组和Adv-GFP组相比,Adv-Mfn2组吸光度值在第3,4,5,6天都显著降低(P<0.01),Adv-Mfn2-PKA(△)组吸光度值无明显变化。与对照组和Adv-GFP组相比,Adv-Mfn2组p-ERK1/2表达水平显著降低(P<0.01),Adv-Mfn2-PKA(△)组无明显变化。提示去除蛋白激酶A磷酸化位点的线粒体融合素2基因定位于线粒体外膜,对血管平滑肌细胞的增殖无拮抗作用,对胞外信号调节蛋白激酶1/2通路无抑制作用。
BACKGROUND: The mitochondrial fusion 2 gene acts on the Ras protein of vascular smooth muscle cells and inhibits cell proliferation through the extracellular signal-regulated protein kinase 1/2 pathway. Serine 442 of the amino acid sequence of mitochondrial fusion 2 gene is a phosphorylation site of protein kinase A, which is closely related to its phosphorylation status and may be involved in its function regulation. OBJECTIVE: To observe the effects of mitochondrial fusion 2 gene on the proliferation of rat vascular smooth muscle cells and its related signal pathways after removing protein kinase A phosphorylation site. Methods: The aortic vascular smooth muscle cells of rats were infected with three kinds of recombinant adenovirus containing the green fluorescent protein gene, mitochondrial fusion 2 gene and mitochondrial fusion 2 gene with the excision of protein kinase A phosphorylation site Subculture 3 to 10 generations were randomized into four groups by lottery: ① without intervention in the control group. ② infected with green fluorescent protein control group (Adv-GFP group). ③ Infection infected mitochondrial fusion 2 gene experimental group (Adv-Mfn2 group). ④ Infection infected mitochondrial fusions 2 gene carrying protein kinase A phosphorylation sites in experimental group (Adv-Mfn2-PKA (△) group). Laser confocal microscopy was used to observe the localization of the mitochondrial fusion 2 gene in the cells, both intact and devoid of protein kinase A phosphorylation sites. Western blot was used to detect the expression of p-ERK1 / 2 and the expression of mitochondrial fusion protein 2 gene in vascular smooth muscle cells, which was intact and removed the phosphorylation site of protein kinase A. MTT method to draw cell growth curve. RESULTS AND CONCLUSION: Mitochondrial fusions 2 gene, both intact and devoid of protein kinase A phosphorylation sites, expressed protein-specific bands in vascular smooth muscle cells. Both gene expression products are mainly distributed in the mitochondrial outer membrane. Compared with Adv-Mfn2-PKA group, the absorbance of Adv-Mfn2 group was significantly decreased on the 3rd, 4th, 5th and 6th days (P <0.01) . The expression of p-ERK1 / 2 in Adv-Mfn2 group was significantly lower than that in Adv-Mfn2-PKA group and Adv-Mfn2-PKA group (P <0.01). It is suggested that the mitochondrial fusion 2 gene, which removes the phosphorylation site of protein kinase A, locates in the mitochondrial outer membrane, has no antagonism on the proliferation of vascular smooth muscle cells and has no inhibitory effect on the extracellular signal-regulated protein kinase 1/2 pathway.