Geminin基因过表达对VSMCs表型转化的影响

Geminin over-expression promotes differentiation in rat vascular smooth muscle cells from dedifferentiation

目的探讨Geminin基因过表达对血管平滑肌细胞(vascular smooth muscle cells,VSMCs)表型转化的影响。方法构建真核表达载体pEGFP-N1-Geminin,脂质体2000介导转染不同表型(去分化型和分化型)的大鼠VSMCs株,设置阳性组、阴性组和空白组(n=3),检测转染效果、VSMCs主要表型标志物(α-actin、OPN)和肌动蛋白相关蛋白1(actinrelated protein,ARP1)的变化情况。结果 Geminin基因成功过表达VSMCs株。与2个对照组(阴性组和空白组)比较,分化型VSMCs中表型标志物(α-actin和OPN)及ARP1含量变化不大;去分化型VSMCs中,OPN在阳性组中表达下调,与2个对照组(阴性组和空白组)比较差异具有统计学意义(P<0.05),α-actin和ARP1在阳性组中表达上调,与2个对照组(阴性组和空白组)比较差异具有统计学意义(P<0.05),免疫共沉淀实验结果提示经protein G plus-Agarose沉淀ARP1相互作用蛋白复合物后,用Geminin抗体进行Western blot检测,可以检测到Geminin的表达。结论 Geminin基因过表达有助于VSMCs由去分化型向分化型转化;免疫共沉淀结果提示Geminin和ARP1之间存在相互作用,ARP1可能参与了上述VSMCs表型转化过程。

Objective To determine the effect of Geminin in the phenotypic transformation of rat vascular smooth muscle cells（VSMCs）.Methods After an eucaryotic plasmid vector pEGFP-N1-Geminin was constructed,VSMCs strains of different phenotypes（dedifferentiated phenotype by cultured in present of serum,and differentiated phenotype by cultured without serum） were transfected with aid of lipofectamine 2000.Cells transfected with pEGFP-N1 and blank plasmids served as negative control and blank control.The expression of Geminin,actin related protein 1（ARP1）,and VSMCs phenotypic makers α-actin and osteopontin（OPN） were detected with Western blotting.The interaction between Geminin and ARP1 were analyzed with co-immunoprecipitation.Results Geminin was successfully overexpressed in VSMCs strains after transfection.Comparing with negative and blank controls,Geminin overexpression resulted in obviously down-regulation of OPN protein in the dedifferentiation cells,but significantly up-regulation in α-actin and ARP1（P0.05）.However,these changes of above-mentioned markers and ARP1 were not significantly changed in differentiated cells.Co-immunoprecipitation indicated that interaction between Geminin and ARP1 was found in Geminin-overexpressed VSMCs strains with dedifferentiated phenotype.Conclusion Geminin may play a role in the phenotypic transformation of VSMCs from dedifferentiation to differentiation.The interaction between Geminin and ARP1 suggests that ARP1 may be involved in this process.