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目的观察线粒体融合蛋白2(Mfn2)si RNA转染人绒毛滋养细胞后自噬的发生情况。方法构建Mfn2 si RNA真核表达载体,电穿孔法将Mfn2 si RNA稳定转染人绒毛滋养层细胞并进行培养,获得Mfn2表达抑制的滋养层细胞。设立实验组(转染Mfn2 si RNA序列)、阴性转染组(转染Cy3 si RNA序列)、空白对照组(未转染序列)。采用Western Blot检测3组细胞转染后Mfn2及自噬相关基因Beclin1和Atg5的表达水平;采用免疫荧光化学染色检测LC3Ⅱ在3组绒毛滋养层细胞中的表达率。结果相对比阴性转染组和空白对照组,实验组Mfn2蛋白水平显著降低,Beclin1和Atg5蛋白表达水平增加;免疫荧光化学染色显示,LC3Ⅱ荧光光点表达率增加。结论 Mfn2低表达诱导Beclin1和Atg5的表达增加,进而诱导绒毛滋养层细胞自噬小体出现和自噬的发生,提示Mfn2可能通过调控绒毛滋养层细胞自噬参与胚胎发育。
Objective To observe the occurrence of autophagy after transfecting human chorionic trophoblasts with mitochondrial fusion protein 2 (Mfn2) si RNA. Methods Mfn2 si RNA eukaryotic expression vector was constructed. The Mfn2 si RNA was stably transfected into human villous trophoblast cells by electroporation, and the trophoblast cells with suppressed Mfn2 expression were obtained. The experimental group (transfected with Mfn2 si RNA sequence), the negative transfected group (transfected with Cy3 si RNA sequence), the blank control group (untransfected sequence) were established. The expression of Mfn2 and autophagy-related genes Beclin1 and Atg5 were detected by Western Blot. The expression of LC3Ⅱ in trophoblast cells was detected by immunofluorescence staining. Results Compared with the negative transfected group and the blank control group, Mfn2 protein level was significantly decreased and Beclin1 and Atg5 protein levels were increased in the experimental group. Immunofluorescence staining showed that the expression rate of LC3 Ⅱ fluorescent spots increased. Conclusion Mfn2 overexpression induced the expression of Beclin1 and Atg5 and induced the appearance of autophagosomes and autophagy in villus trophoblast cells, suggesting that Mfn2 may be involved in embryonic development by regulating the autophagy of villus trophoblast cells.