转染miR-17-5p对PC12细胞Bcl-2、Bax蛋白表达和形态学影响

Effects of miR-17-5p transfection on the expression of Bcl-2 and Bax protein and morphology of PC12 cells

目的:观察基因miR-17-5p转染对PC12细胞Bcl-2、Bax蛋白表达和形态学影响。方法:用质粒PEGFP-N1-vector空载体和PEGFP-N1-miR-17-5p表达载体转染正常的PC12细胞,将细胞分为对照组、空载组、miR-17-5p组。应用免疫细胞化学染色法和Western-blot方法检测凋亡相关蛋白Bcl-2、Bax的表达,电镜观察各组细胞超微结构改变。结果:对照组与空载组Bcl-2、Bax蛋白表达无显著性差异,miR-17-5p组与空载组比较,Bcl-2蛋白表达明显减少(P<0.01),而Bax蛋白表达明显增多(P<0.01),Western blot检测其凋亡相关蛋白表达变化与免疫组化结果一致。电镜下观察可见正常对照组与空载组细胞形态无明显差异,细胞形态结构正常,无明显病理改变;miR-17-5p转染组细胞胞体肿胀,线粒体髓样变和空泡样变,胞核扭曲凹陷,异染色质边集。结论:miR-17-5p基因过表达能引起PC12细胞促凋亡的Bax蛋白表达增多,抗凋亡的Bcl-2蛋白表达减少,导致PC12细胞形态学损伤。

Objective： To observe the effect of miR-17-5p gene transfection on the expression of Bcl-2 and Bax protein and morphology of PC12 cells. Methods： PC12 cells were transfected with PEGFP-N1-vector and PEGFP-N1-miR-17-5p plasmids. Cells were divided into three groups： control group,unladen group and miR-17-5p group. Using immunocytochemical and Western blot method to detect the expression of Bcl-2,Bax protein in PC12 cell. The changes of ultrastructure of cells were detected by electron microscopy. Results： There was no significant difference between the control group and the unladen group in the expression of Bcl-2 and Bax protein. Compared with control group,expression of Bcl-2 protein in miR-17-5p group was significantly decreased（ P〈0. 01）,and the expression of Bax protein was significantly increased（ P〈0. 01）. Western blot results were in agreement with immunocytochemical results. The electron microscope results showed,cells in the unladen group and the control group had normal morphology and no obviously pathological changes were observed. The cells of the miR-17-5p transfected groups were slightly big with distorted and depressed and heterochromatin marginalized,mitochondria had medullary and vacuolation change nuclei. Conclusion： miR-17-5p over-expression can induce down-regulation of Bcl-2 protein and up-regulation of Bax protein,causing the injury of cytomorphology.