摘要
目的 :通过观察 c- Src在 Ang 对大鼠血管平滑肌细胞 (VSMC)丝裂原活化的蛋白激酶 (MAPK)活性和c- fos蛋白表达的影响 ,以进一步了解 Ang 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/ L Ang 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 ;Western blot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 ,0 .2 μmol/ L、0 .5 μmol/ L、1.0 μmol/ L和 2 .0 μmol/ L分别为对照的 6 8.2 %、34.7%、30 .3%和 15 .8% ,经方差分析具有显著性意义 (P<0 .0 1)。c- Src激酶活性也显著抑制 ;以 Ang 刺激经转染反义 c- Src ODNs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 :Ang 可诱导 VSMC c- Src激活和细胞内信息转导 ,且 Ang 引起的 MAPK和 c-
Objective: To further clarify the mechanism of AngⅡ induced intracellular signal transduction in vascular smooth muscle cells(VSMCs) proliferation by observing the effect of c Src on AngⅡ mediated MAPK activation and c fos protein expressions in rat VSMCs. Methods: Aortic VSMCs from SD rats were cultured primarily and subcultured, which were transfected with anti sense c Src oligodeoxynucleotides(ODNs) wrapped with lipofectin to inhibit c Src activity and protein production. Untransfected VSMCs were used as control, to observe the role of AngⅡ stimulation in MAPK activation and c fos protein expression in VSMC. Protein immunoprecipitation and kinase phosphorylation were employed to measure c Src kinase activity; MAPK kinase activity was assessed by the phosphorylation rate of the substrate MBP (Myelin Basic Protein); Western blot was used to assess the protein expression of c Src and c fos. Results: c Src protein expressions in VSMC, which were transfected with different concentrations of anti sense c Src ODNs, were significantly decreased in a negative dose effect manner (0.2 μm, 0.5 μm, 1.0 μm and 2.0 μm were 68.2%, 34.7%, 30.3% and 15.8% respectively compared with control). c Src kinase activity was also obviously inhibited. Following stimulation of AngⅡ on VSMC transfected with anti sense c Src ODNs, the increase of c Src activity was only 8.7% of control,the activity of MAPK only 1.6% compared with control, and the increase in c fos protein expression 30.3% as control. Conclusion: AngⅡ can induce c Src activation and intracellular signal transduction in VSMC which depend on c Src activation, indicating that c Src is a pivotal signal factor in VSMC proliferation.[
出处
《浙江大学学报(医学版)》
CAS
CSCD
2003年第2期126-130,共5页
Journal of Zhejiang University(Medical Sciences)
基金
国家自然科学基金资助项目 (39770 319)