摘要
目的 探讨胰岛素样生长因子 1(IGF 1)促血管平滑肌细胞 (VSMC)增殖的细胞内信号转导机制及雷帕霉素干预作用。 方法 兔血管平滑肌细胞分 6组处理 ,以细胞计数、噻唑盐比色法测定细胞增殖能力 ,WesternBlot测定蛋白激酶B (PKB)表达水平 ,免疫沉淀、特异底物组蛋白H2Bγ3 2 P掺入量测定PKB活性。以磷脂酰肌醇 3激酶 (PI3K)特异性抑制剂渥漫青霉素处理细胞间接反映PI3K作用。 结果 IGF 1使细胞增殖增加至 2 8～ 3 8倍 ,IGF 1对PKB活性的影响呈浓度 (1～ 10 0 μg/L)依赖性增加及时间 (10min～ 2 4h)依赖性降低 ,1μg/L刺激 10min即使PKB活性增至 2倍 ,10 0 μg/L时增至近 6 0倍 ,10 0 μg/L刺激 2 4hPKB活性增加仍达 4倍以上。雷帕霉素在浓度为 10～ 10 0nmol/L范围内使PKB活性进行性降低 2 5 %～ 73% ,渥漫青霉素和雷帕霉素使VSMC增殖至少降低 30 % ,并完全逆转IGF 1的上述作用。 结论 雷帕霉素可抑制IGF 1促VSMC增殖及对生长信号PI3K/PKB的活化。
Objective To investigate the intracellular signal transduction pathway for the vascular smooth muscle cell (VSMC) proliferation stimulated by insulin like growth factor 1(IGF 1) and the intervention effect of rapamycin. Methods Rabbit aortic VSMCs were cultured in 6 groups. Cell proliferating ability was determined by measuring cell number and mitochondrial dehydrogenase activity(MTT assay).Western blotting was used to detect the expression of protein kinase B(PKB).Immunoprecipitation and radioactivity of γ 32 P incorporated into its specific substrate histon H2B were utilized to evaluate PKB activity. Wortmannin(WT),the specific inhibitor of phosphatidylinositol 3 kinase(PI3K), was used to reflect indirectly the possible involvment of PI3K. Results IGF 1(100 μg/L) increased cell number and mitochondrial dehydrogenase activity to 2 8～3 8 fold. IGF 1 elevated PKB activity in a concentration dependent manner (1～100 μg/L),with 1 0～58 9 times increase,and in time dependent manner (10 min～24 h) , from 58 9 times at 10 min to 4 47 times at 24 h respectively.Rapamycin (10～100 nmol/L) decreased the PKB activity by 25%～73% in a concentration dependent manner. Both WT and rapamycin markedly lowered VSMC proliferation by more than 30% and completely abolished the above effects of IGF 1. Conclusions It is suggested that rapamycin may inhibit PI3K/PKB signal transduction pathway involved in rabbit VSMC proliferation stimulated with or without IGF 1.
出处
《中华老年医学杂志》
CAS
CSCD
北大核心
2003年第4期233-236,共4页
Chinese Journal of Geriatrics
