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目的 探讨氧化低密度脂蛋白 (oxLDL)促血管平滑肌细胞 (VSMC)增殖及分泌功能改变的细胞内信号转导机制及雷帕霉素 (rapamycin)干预作用。方法 培养兔血管平滑肌细胞分为 7组处理。以直接细胞计数及噻唑盐 (MTT)比色法测定细胞增殖能力 ;酶联免疫法检测肿瘤坏死因子α(TNF α)、白细胞介素 6 (IL 6 )、白细胞介素 8(IL 8)水平 ;Western免疫印迹法定量蛋白激酶B(PKB)表达水平 ;免疫沉淀、特异底物组蛋白H2 Bγ32 P掺入量测定PKB活性。结果 oxLDL使细胞增殖指标增加 1.6 2～ 3 .2倍 ,细胞因子分泌增加 3～ 5倍 ,PKB活性增加 11.8倍。而相同浓度的低密度脂蛋白对细胞增殖及细胞因子分泌无明显影响。磷脂酰肌醇 3 激酶 (PI3K)特异性抑制剂沃漫青霉素 (Wortman nin ,WT)及雷帕霉素均可显著抑制VSMC增殖、细胞因子分泌及PKB活性 ,并完全逆转oxLDL的上述作用。结论 PI3K PKB是oxLDL促VSMC增殖及分泌功能的信号通路之一。雷帕霉素可能通过抑制PI3K PKB对VSMC生物学功能发挥较全面调控效用。
Objective To investigate the mechanism of intracellular signal transduction and the effect of rapamycin on the proliferation and secretion of oxidized low density lipoprotein (oxLDL) -stimulated vascular smooth muscle cells (VSMCs). Methods Rabbit vascular smooth muscle cells were divided into 7 groups. The cell proliferation was measured by direct cell counting and MTT colorimetric assay. The levels of tumor necrosis factor α (TNFα), interleukin 6 (IL 6) and interleukin 8 (IL 8) Western blotting was used to determine the protein kinase B (PKB) expression. Immunoprecipitation and specific substrate histone H2 Bγ32 P incorporation were used to determine PKB activity. Results oxLDL increased cell proliferation index by 1.6 2 ~ 3.2 times, cytokine secretion increased 3 ~ 5 times and PKB activity increased 11.8 times. The same concentration of low density lipoprotein on cell proliferation and cytokine secretion had no significant effect. Both phosphatidylinositol 3-kinase (PI3K) specific inhibitor Wortman nin (WT) and rapamycin significantly inhibited VSMC proliferation, cytokine secretion and PKB activity, and completely reversed the effect of oxLDL. Conclusion PI3K PKB is one of the signaling pathways of oxLDL promoting proliferation and secretion of VSMCs. Rapamycin may exert a more comprehensive regulatory effect on the biological function of VSMC by inhibiting PI3K PKB.