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目的:探讨线粒体融合素2(Mfn2)基因蛋白激酶A(PKA)磷酸化位点突变对大鼠颈动脉球囊损伤后血管平滑肌细胞凋亡的影响。方法:建立大鼠颈动脉球囊损伤模型,感染组分别以携带全长Mfn2基因的重组腺病毒(Adv-Mfn2)、携带不同Mfn2基因PKA磷酸化位点突变体的重组腺病毒(Adv-Mfn2-S442A,Adv-Mfn2-S442D)和仅含有半乳糖苷酶基因的对照腺病毒(Adv-LacZ)感染损伤的颈总动脉段,以注射磷酸盐缓冲液(PBS)作为未感染对照组,同时以假手术组作为正常对照组,分别于术后5 d、14 d取材,应用免疫组化检测外源基因的表达,原位末端转移酶标记法(TUNEL)检测凋亡细胞,苏木精-伊红染色行组织形态学分析。结果:术后5 d,免疫组化证实Adv-Mfn2、Adv-Mfn2-S442A、Adv-Mfn2-S442D 3组Mfn2蛋白表达水平明显增加,TUNEL染色显示Adv-Mfn2、Adv-Mfn2-S442A组凋亡细胞数较对照组、Adv-LacZ组和Adv-Mfn2-S442D组明显增加(P<0.01);与Adv-Mfn2组相比,Adv-Mfn2-S442A组凋亡细胞数更多(P<0.01)。术后14 d,苏木精-伊红染色显示Adv-Mfn2组、Adv-Mfn2-S442A组内膜/中膜面积比明显低于对照组、Adv-LacZ组和Adv-Mfn2-S442D组(P<0.01);且Adv-Mfn2-S442A组内膜/中膜面积比值较Adv-Mfn2组更低(P<0.01)。结论:高表达Mfn2基因可促进大鼠颈动脉球囊损伤后血管平滑肌细胞凋亡,且Mfn2-S442A促凋亡作用更强,而Mfn2-S442D无此作用。
OBJECTIVE: To investigate the effect of PKF phosphorylation site mutation of mitochondrial fusion 2 (Mfn2) gene on vascular smooth muscle cell apoptosis after carotid artery balloon injury in rats. Methods: The model of carotid artery balloon injury in rats was established. The infected mice were respectively infected with recombinant adenovirus (Adv-Mfn2) carrying full-length Mfn2 gene and Adv-Mfn2 carrying different Mfn2 gene PKA phosphorylation sites S442A, Adv-Mfn2-S442D) and control adenovirus containing only the galactosidase gene (Adv-LacZ) were injected into the common carotid artery segment to inoculate phosphate buffered saline (PBS) into the uninfected control group The sham-operation group was taken as the normal control group. The rats were sacrificed on the 5th and 14th day respectively. The expression of the foreign gene was detected by immunohistochemistry. The apoptotic cells were detected by TUNEL, Eosin staining histomorphology analysis. Results: The expression of Mfn2 protein in Adv-Mfn2, Adv-Mfn2-S442A and Adv-Mfn2-S442D3 groups was significantly increased 5 days after operation. TUNEL staining showed apoptosis in Adv-Mfn2 and Adv-Mfn2-S442A groups Compared with Adv-Mfn2 group, the number of apoptotic cells in Adv-Mfn2-S442A group was more than that in Adv-Mfn2-S442A group (P <0.01) . At 14 days after operation, the intima / media area ratio in Adv-Mfn2 group and Adv-Mfn2-S442A group was significantly lower than that in control group by hematoxylin-eosin staining. Adv-LacZ group and Adv-Mfn2-S442D group <0.01). The intima / media area ratio of Adv-Mfn2-S442A group was lower than Adv-Mfn2 group (P <0.01). CONCLUSION: Mfn2 gene can promote the apoptosis of vascular smooth muscle cells after carotid artery balloon injury in rats, and Mfn2-S442A has a stronger pro-apoptotic effect than Mfn2-S442D.