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目的:本研究通过观察c -Src在AngII对大鼠血管平滑肌细胞(VSMC)丝裂原激活的蛋白激酶(MAPK)的激活和c -Fos蛋白表达中的影响,以进一步阐明AngII促VSMC增殖的细胞内信息转导机制。方法:原代和传代培养SD大鼠主动脉VSMC ,以脂质体包裹反义c -Src寡脱氧核苷酸(oligodeoxynucleotides,ODNs)转染培养的VSMC以抑制c -Src蛋白表达和激酶活性。以未转染的VSMC为对照,观察10 -7mol/LAngII刺激对转染的VSMC的MAPK活性和c -Fos蛋白表达的影响。蛋白免疫沉淀和酶自身磷酸化率法测定c -Src激酶活性;髓鞘碱性蛋白(MBP)底物磷酸化率测定MAPK激酶活性;Westernblotting免疫印迹法测定c-Src和c -Fos蛋白表达情况。结果:转染不同浓度反义c-SrcODNs的VSMC ,c -Src蛋白含量呈浓度依赖性降低,c-Src激酶活性也显著抑制,以AngII刺激经转染反义c -SrcODNs的VSMC ,c -Src激酶活性增幅仅为对照组的8 .7% ;MAPK活性仅为对照的1 .6 % ;c -Fos蛋白表达的增幅为对照组的30 .0 %。结论:AngII可诱导VSMCc -Src激活和细胞内信息转导,且AngII引起的MAPK和c -fos的激活依赖于c-Src的激活,提示c -Src是AngII促血管平滑肌细胞增殖的重要信息分子。
Objective: To investigate the effect of c-Src on the activation of mitogen-activated protein kinase (MAPK) and the expression of c-Fos protein in rat vascular smooth muscle cells (VSMCs) in order to further elucidate the role of Ang II in VSMC proliferation Intracellular information transduction mechanism. Methods: VSMCs of aorta of SD rats were subcultured in primary and secondary cultures and VSMCs were transfected with antisense c-SCR oligodeoxynucleotides (ODNs) to inhibit the expression of c-Src protein and kinase activity. The untransfected VSMC was used as a control to observe the effects of 10 -7 mol / LAngII stimulation on MAPK activity and c-Fos protein expression in transfected VSMCs. C-Src kinase activity was measured by protein immunoprecipitation and enzyme autophosphorylation assay; MAPK kinase activity was measured by phosphorylation of MBP substrate; c-Src and c-Fos protein expression was determined by Western blotting . Results: The VSMCs with different concentrations of antisense c-SrcODNs transfected with antisense c-SrcODNs showed a concentration-dependent decrease in c-Src kinase activity and c-Src kinase activity. VSMCs transfected with antisense c-SrcODNs and c- Src kinase activity increased only 8.7% of the control group; MAPK activity was only 1.6% of the control; c-Fos protein expression increased 30.0% of the control group. CONCLUSION: AngII can induce VSMCc-Src activation and intracellular signal transduction. The activation of MAPK and c-fos induced by AngII depends on the activation of c-Src, suggesting that c-Src is an important information molecule for the proliferation of AngII-stimulated vascular smooth muscle cells .