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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 investigate the effect of Geminin gene overexpression on phenotypic transformation of vascular smooth muscle cells (VSMCs). Methods The eukaryotic expression vector pEGFP-N1-Geminin was constructed and transfected into rat VSMCs with different phenotypes (dedifferentiated and differentiated) by lipofectamine 2000. Positive cells, negative cells and blank group (n = 3 ), The transfection effect, the changes of the main phenotype markers (α-actin, OPN) and actin-related protein 1 (ARP1) of VSMCs were detected. Results The Geminin gene overexpression VSMCs strain. Compared with the two control groups (negative group and blank group), the expression of phenotype markers (α-actin and OPN) and ARP1 did not change much in differentiated VSMCs. In dedifferentiated VSMCs, OPN expression was down-regulated in the positive group, (P <0.05). The expressions of α-actin and ARP1 were up-regulated in the positive group compared with the two control groups (negative group and blank group), the difference was statistically significant (P <0.05). The results of co-immunoprecipitation suggested that Geminin could be detected by Western blot with Geminin antibody after protein G plus-Agarose precipitated ARP1-interacting protein complex. Conclusion Geminin overexpression contributes to VSMCs transformation from dedifferentiation to differentiation. Immunoprecipitation results suggest that there is interaction between Geminin and ARP1, and ARP1 may be involved in phenotypic transformation of VSMCs.