PCSK9介导ApoER2降解在血管平滑肌细胞多倍体化及衰老中的作用

Role of PCSK9 Mediating ApoER2 Degradation in Polyploidization and Senescence of Vascular Smooth Muscle Cells

目的观察PCSK9过表达对人血管平滑肌细胞多倍体化及衰老的影响,并探讨ApoER2在其过程中发挥的作用。方法空载腺病毒(Ad-Null)和PCSK9过表达腺病毒(Ad-PCSK9)转染人血管平滑肌细胞,光镜观察细胞形态学改变;采用BrdU/PI染色,并采用LSC法检测细胞的增殖及多倍体水平;采用β-半乳糖苷酶检测试剂盒检测细胞衰老情况;采用Western blot法检测PCSK9和ApoER2的表达水平。采用人重组ApoER2蛋白(rhApoER2)干预Ad-PCSK9转染的人血管平滑肌细胞,并检测其增殖及多倍体水平。结果与Ad-Null转染组比较,光镜下Ad-PCSK9转染组中多倍体血管平滑肌细胞的数目显著增加(P<0.01);LSC法检测结果显示,Ad-PCSK9转染组的细胞多倍体水平显著上调并且增殖活性明显下降(P<0.01);β-半乳糖苷酶检测结果显示,PCSK9转染组中SAβG阳性细胞数目显著增加(P<0.01);Western blot法检测结果显示,ApoER2的表达水平随Ad-PCSK9的过表达而显著下调(P<0.01)。LSC法检测结果显示,rhApoER2可以显著抑制Ad-PCSK9诱导的多倍体血管平滑肌细胞形成(P<0.05)。结论PCSK9可通过介导人血管平滑肌细胞ApoER2的降解,促进血管平滑肌细胞多倍体的形成及衰老。

Objective To observe the effect of PCSK9 on the polyploidization and senescence of human vascular smooth muscle cells(VSMCs),and to explore the role of ApoER2 in this process.Methods The VSMCs were transfected with Ad-Null and PCSK9-overexpressing adenovirus(Ad-PCSK9),and light microscope was used to observe the morphological changes of the cells.BrdU/PI staining and LSC method were used to detect cell proliferation and polyploidization level.Theβ-galactosidase assay kit was used to determine the senescence of cells.Western blot was used to detect the expression levels of PCSK9 and ApoER2.Recombinant human ApoER2(rhApoER2)was used for the intervene VSMCs transfected with Ad-PCSK9,and the level of proliferation and polyploidization were detected.Results Compared with the Ad-Null transfection group,the number of polyploid VSMCs were significantly increased in Ad-PCSK9 transfected group under light microscope(P<0.01).LSC assay showed that the polyploid level of Ad-PCSK9 transfected group was significantly up-regulated and the proliferative activity significantly decreased(P<0.01).β-galactosidase assay showed that the number of SAβG-positive cells were significantly increased in Ad-PCSK9 transfected group(P<0.01).Western blot analysis showed that the expression level of ApoER2 was significantly down-regulated with the overexpression of Ad-PCSK9(P<0.01).LSC assay showed that rhApoER2 significantly inhibits the formation of polyploid VSMCs induced by Ad-PCSK9(P<0.05).Conclusion PCSK9 can promote the formation of polyploidization and senescence of VSMCs by mediating the degradation of ApoER2.