曲古菌素A对血管平滑肌细胞p27kip1表达的调控机制研究

Effect of Trichostatin A on p27kip1 Expression in Vascular Smooth Muscle Cells

目的探讨组蛋白去乙酰化酶抑制剂曲古菌素A(Trichostatin A,TSA)对血管平滑肌细胞(VSMC)的p27kip1表达的影响和调控机制。方法半定量逆转录聚合酶链反应(RT-PCR)检测p27kip1的mRNA水平,蛋白印迹测定p27kip1和S-phase kinase-associated protein-2(skp2)蛋白表达,荧光分光光度法测定20S蛋白酶体活性。结果100 ng/ml TSA不影响VSMC中p27kip1的mRNA水平。100 ng/mlTSA显著抑制血清诱导的p27kip1蛋白下调,并延长p27kip1蛋白的半衰期。100 ng/ mlTSA抑制血清诱导的skp2表达上调,且skp2表达与相应时点p27kip1蛋白呈负相关。100 ng/ml TSA对20S蛋白酶体活性物影响。结论TSA对VSMC的p27kip1表达调控不是在转录水平上,而是通过翻译后机制抑制血清诱导VSMC的p27kip1蛋白降解,其机制可能与TSA抑制泛素连接酶亚单位skp2表达有关。

Objective To investigate the effect of Trichostatin A （TSA） on p27kip1 gene expression in vascular smooth muscle ceils. Methods Reverse Transcription-Polymerase Chain Reaction was used to measure the level of p27kip1 mRNA. The levels of p27kip1 and S-phase kinase-associated protein-2 （ skp2 ） protein were determined by western blotting. 20S proteasome activity was quantified by using a fluorogenic proteasome-specific substrate. Results TSA did not affect mRNA level of p27kip1 in VSMC, but attenuated serum-induced downregulation of p27kip1 through stabilizing p27kip1 turnover. In addition, TSA potently decreased the expression of skp2, an F-box protein that targets p27kip1 for degradation, but had no effect on protease activity. Conclusion TSA regulate p27kip1 expression at the post-translational level in VSMC.