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目的通过比较静止状态的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)与血清刺激后的VSMCs在分化表型改变中Geminin的表达变化情况,探讨Geminin对VSMCs表型转化的影响。方法实验设置静止状态(血清饥饿)对照组、血清刺激组,研究不同时相两个组Geminin的分布、活性及表达水平的差异。血清饥饿法可以诱导出VSMCs表型分化及活性差异;免疫荧光染色后激光共聚焦显微镜观察Geminin核内定位;应用PCR检测Geminin基因表达的变化;Western blot检测Geminin蛋白、VSMC合成型标志物骨桥蛋白(osteopontin,OPN)及收缩型标志物平滑肌肌动蛋白相关蛋白(actin-related proteins,ARP)表达的变化。结果①静止状态下(血清饥饿)VSMCs以收缩型为主,血清刺激后以合成型为主;②静止状态Geminin几乎全部存在于细胞质内,而血清刺激后Geminin几乎全部定位于胞核内;③GemininmRNA在血清刺激时明显升高,且在刺激48h时差异表达最明显,与无血清比较差异具有统计学意义(P<0.05);④Geminin蛋白和骨桥蛋白在血清刺激时均明显升高,与无血清比较差异具有统计学意义(P<0.05)。结论 Geminin参与介导了VSMCs由收缩型向合成型的转化。
OBJECTIVE: To investigate the effect of Geminin on the phenotypic changes of VSMCs by comparing the changes of Geminin expression in VSMCs with quiescent VSMCs and serum VSMCs. Methods The rats in the quiescent state (serum starvation) control group and the serum stimulation group were set up to study the distribution, activity and expression level of Geminin in two groups at different time points. Serum starvation method can induce VSMCs phenotypic differentiation and activity difference; immunofluorescence staining confocal laser scanning microscope Geminin nuclear localization; Geminin gene expression changes detected by PCR; Western blot detection of Geminin protein, VSMC marker synthetic bone bridge (Osteopontin, OPN) and contractile marker smooth muscle actin-related proteins (ARP). Results (1) VSMCs were mainly contracted in resting state (serum starved) and mainly in syncytial form stimulated by serum; (2) Geminin almost remained in the cytoplasm at rest, while almost all Geminin localized in the nucleus after serum stimulation; (3) Geminin mRNA (P <0.05) .④Geminin protein and osteopontin were significantly increased when stimulated with serum, but not significantly different from those without serum Serum differences were statistically significant (P <0.05). Conclusion Geminin participates in the transformation of VSMCs from contractile to synthetic.