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目的:研究Rapamycin(Rapa)阻断血管紧张素Ⅱ(AngⅡ)刺激血管内皮细胞增生信号传导机制。方法:不同浓度AngⅡ刺激人脐静脉内皮细胞(ECV304),并用Rapa进行干预,采用3H胸腺嘧啶核苷掺入法和3H亮氨酸掺入法测定细胞DNA和蛋白质合成,流式细胞术检测细胞周期变化,免疫印迹(Westernblot)检测细胞信号蛋白P70s6k,ERK2及细胞周期蛋白CyclinD1、CyclinA、和CyclinB1表达的变化。结果:AngⅡ刺激细胞24h。细胞DNA和蛋白质合成均增加,并呈剂量依赖效应。AngⅡ10-6M组与对照组比较P70s6k,ERK2表达分别上调108.7%,54.7%,Rapa完全阻断P70s6k和CyclinD1的活化,而不影响ERK2的表达。结论:Rapa通过阻断PI3Kp70S6K信号通路抑制AngⅡ诱导的血管内皮细胞增生,抑制细胞周期蛋白CyclinD1的表达,阻止细胞G0/G1→S期。
AIM: To investigate the mechanism of rapamycin (Rapa) blocking angiotensin Ⅱ (AngⅡ) - stimulated proliferation of vascular endothelial cells. Methods: Human umbilical vein endothelial cells (ECV304) were stimulated with different concentrations of AngⅡ and treated with Rapa. DNA and protein synthesis were determined by 3H thymidine incorporation and 3H-leucine incorporation. Flow cytometry The changes of cell signal proteins P70s6k, ERK2 and cyclinD1, CyclinA, and CyclinB1 were detected by Western blot. Results: Ang Ⅱ stimulated cells 24h. Cell DNA and protein synthesis increased, and in a dose-dependent manner. AngⅡ10-6M group compared with the control group P70s6k, ERK2 expression increased 108.7%, 54.7%, Rapa completely blocked P70s6k and CyclinD1 activation, without affecting ERK2 expression. Conclusion: Rapa can inhibit the proliferation of vascular endothelial cells induced by AngⅡ, block the expression of CyclinD1 and block the G0 / G1 → S phase by blocking PI3Kp70S6K signaling pathway.