摘要
目的研究血管紧张素Ⅱ(AngⅡ)对血管平滑肌细胞Cav3.1表达的影响及其作用机制。方法酶消化法原代培养大鼠主动脉血管平滑肌细胞,应用第5代传代细胞,实验随机分为7组:对照组(不加任何药物)、AngⅡ组[加入AngⅡ(0.1μmol/L)培养24 h]、AngⅡ+氯沙坦钾组[加入氯沙坦钾(1μmol/L)培养1 h,再加入AngⅡ(0.1μmol/L)共同培养24 h]、AngⅡ+PD98059组[加入PD98059(10μmol/L)预刺激1 h,再加入AngⅡ(0.1μmol/L)共同培养24 h]、PD98059组[加入PD98059(10μmol/L)培养1 h]、AngⅡ+氯沙坦钾+PD98059组[先加入氯沙坦钾(1μmol/L),PD98059(10μmol/L)培养1 h,再加入AngⅡ(0.1μmol/L)共同培养24 h]、氯沙坦钾组[加入氯沙坦钾(1μmol/L)培养4 h]。RT-PCR、Western blot方法测定各组T型钙通道的表达。结果PCR结果显示:与对照组(1.000±0.315)比较,AngⅡ组Cav3.1表达量(17.930±0.954)明显增加(P<0.01);PD98059组、氯沙坦钾组Cav3.1表达量为0.928±0.262、0.978±0.292,与对照组比较差异无统计学意义(P>0.05);AngⅡ+氯沙坦钾组、AngⅡ+PD98059组、AngⅡ+氯沙坦钾+PD98059组的Cav3.1表达量分别为0.125±0.007、0.066±0.015、0.109±0.018,明显低于AngⅡ组(P<0.01)。Western blot结果显示:与对照组比较,AngⅡ+氯沙坦钾组、AngⅡ+PD98059组及AngⅡ+氯沙坦钾+PD98059组Cav3.1蛋白表达明显降低(P<0.05);而PD98059组及氯沙坦钾组Cav3.1蛋白表达与对照组比较差异无统计学意义(P>0.05)。结论 AngⅡ通过Ras/PKCζ/MEK/ERK1/2路径增加血管平滑肌细胞Cav3.1的表达。
Objective To investigate the effects of Ang Ⅱ on T-type calcium channels in rat vascular smooth muscle cells. Methods Primary. cul- ture of rat aortic vascular smooth muscle cells was established by enzyme digestion method, and the fifth generation cells were used in the study. Cells were randomly divided into 7 groups : ①control group : without any drugs ; ②Ang Ⅱ group : Ang Ⅱ 0.1 μmol/L for 24 hours ; ③Ang Ⅱ +losartan po- tassium group: losartan potassium 1 μmol/L for 1 hour, then add Ang Ⅱ 0.1 μmol/L and cultures for another 24 hours ; ④Ang Ⅱ +PD98059 group : adds 10 μmol/L PD98059 and stimulus for 1 hour, then adds 0.1 μmol/L Ang Ⅱ and cultures for 24 hours together; ⑤PD98059 group : adds 10 μmol/L PD98059 and cultures for 1 hour; ⑥Ang Ⅱ + losartan potassium + PD98059 group: adds 1 μmol/L losartan potassium and 10 μmol/L PD98059 cultures for 1 hour, then adds 0.1 μmol/L Ang Ⅱ and cultures for 24 hours together; ⑦losartan potassium group : adds losartan potassium and cultures for 4 hours. The expression of T-type calcium channel of every group was determined by PT-PCR and Western blot. Results ①PCR results showed that the expression of Cav3.1 in Ang Ⅱ group ( 17.930±0.954 ) increased significantly compared with control group (1.000±0.315 ) (P 〈 0.05 ), which were not observed in PD98059 group (2^-△△Ct : 0.928±0.262 ) and losartan potassium group (2^-△△Ct: 0.978±0.292) ; the expression of Cav3.1 in Ang Ⅱ +losartan potassium group (0.125±0.007 ), Ang Ⅱ +PD98059 group (0.066±0.015 ) and Ang Ⅱ + losartan potassium + PD98059 group (0.109±0.018) significantly lower than Ang Ⅱ group ( 1.000±0.053 ) (all P 〈 0.01 ).②Western blot result showed that the expression of Cav3.1 in Ang Ⅱ +losartan potassium group, Ang Ⅱ + losartan potassium + PD98059 group and Ang Ⅱ +PD98059 group is significantly reduced compared with the control group ; while no obvious statistical significance was found between PD98059 group and losartan potassium group (P 〉 0.05 ). Conclusion Angiotensin Ⅱ can lead excessive expression of T-type calcium channel in rat vascular smooth muscle cells ; However, there is no statistically significant differencescompamd with the control group.
出处
《中国医科大学学报》
CAS
CSCD
北大核心
2014年第9期802-805,共4页
Journal of China Medical University
基金
辽宁省教育厅高校科研计划(L2013314)