线粒体融合素2基因突变体对大鼠颈动脉球囊损伤后内膜增生的影响

目的 探讨线粒体融合素2(mitofusin 2,Mfn 2)基因蛋白激酶A(PKA)磷酸化位点突变体对大鼠颈动脉球囊损伤后内膜增生的影响.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.术后14 d取材,采用HE染色、Image-Pro plus图像分析系统进行形态学分析,同时,应用免疫组化检测Mfn2蛋白和细胞增殖核抗原(PCNA)表达,应用方差分析和Dunnett-t检验行统计学分析.结果 术后14 d,免疫组化证实Adv-Mfn2、Adv-Mfn2-S442A、Adv-Mfn2-S442D组Mfn2总蛋白表达水平明显增加;HE染色和PCNA染色结果显示,Adv-Mfn2组、Adv-Mfn2-S442A组内膜/中膜面积比(I/M)和PCNA阳性细胞率明显低于Control组(P＜0.01);与Adv-Mfn2组相比,Adv-Mfn2-S442A组I/M值和PCNA阳性细胞率更低(P＜0.01);Aay-LdacZ组和Adv-Mfn2-S442D组与Control组相比差异无统计学意义(P＞0.05).结论 高表达Mfn2基因可抑制大鼠颈动脉球囊损伤后内膜增生,且Mfn2基因突变体Mfn2-S442A抑制作用更强,而Mfn2-S442D则无抑制作用.

The effects of mutation of PKA phosphorylation site of Mfn2 on the intimal proliferation after balloon injury of carotid artery in rats

Objective To study the effects of protein kinase A (PKA) phosphorylation site' s mutation of mitofusin-2 (Mfn2) on intimal proliferation after balloon injury of carotid artery of rats. Method Rat model of carotid artery balloon injury was established and infected with Adv-LacZ, Adv-Mfn2, AdvMfn2-S442A or Adv-Mfn2-S442D from the peri-arterial sheathes of vessels, while phosphate buffered solution (PBS) used instead of above infectious adenovirus as uninfected group and sham operation as control group. The rats were sacrificed 14 days after balloon injury of carotid artery in order to measure the level of Mfn2 protein and the prol9iferation cell nuclear antigen (PCNA) with immunohistochemistry staining. The morphology of vessels was observed with HE staining. All data were statistically analyzed with ANOVA and Dunnett-t test. Results Fourteen days after surgery, the levels of Mfn2 protein significantly increased in arteries infected with Adv-Mfn2, Adv-Mfn2-S4442A and Adv-Mfn2-S442D compared with those in control group, sham operation group and Adv-LacZ infected group. The ratio of intimal area/medial area (I/M) and percentage of PCNA positive cells in both Adv-Mfn2 and Adv-Mfn2-S442A groups markedly decreased compared with control group (P ＜0. 01 ) . Compared with the Adv-Mfn2 group, the I/M and the percentage of PCNA positive cells reduced more significantly in Adv-S442A group (P ＜ 0. 01 ) . However,the I/M and the percentage of PCNA positive cells in Adv-LacZ and Adv-S442D groups were not significantly different from those found in the control group. Conclusions The over-expression of Mfn2 gene may effectively inhibit intimal proliferation after balloon injury of carotid artery of rats. The inhibitory effects of Mfn2-S442A are more obvious than those of Mfn2. However, the Mfn2-S442D is out of the inhibitory effect on neo-intimal proliferation.