转染血管紧张素受体Ⅱ(AT_l)反义核苷酸对血管平滑肌细胞增殖的影响

Effect of Transfecting Angiotensin Ⅱ Receptor(AT 1) Antisense \;Nucleotide on the Growth of Vascular Smooth Muscle Cells

目的 :评价转染 ATl 反义核菩酸 (ATl A)对血管平滑肌细胞(VSMCs)血管紧张 (Ang )受体亚型 m RNA表达、蛋白激酶 C(PKC)、丝裂素活化蛋白激酶 P38(P38MAPK)蛋白表达 ,及蛋白核酸合成的作用。方法 :RT- PCR克隆 ATl c DNA序列 (476 bp) ,将克隆的 ATlc DNA反向插入 Pc DNA3.1 ,构建一完整的含 ATl A的质粒(PATl A) ,测序鉴定。转染培养的大鼠 VSMCs,RT- PCR检测转染 VSMCs AT1 m RNA表达。Ang (1 0 - 7mol/ L)剌激 2 4h后 ,比较转染与非转染的 VSMCs AT1 与 AT2 m RNA表达(RT- PCR)、P38MAPK和 PKC蛋白表达 (免疫印迹 ,westernblot)、蛋白核酸合成 (3H- L eucine及 3H- Thym idine掺入 )。结果 :成功构建 PATl A。RT- PCR显示转染 VSMCs ATlm RNA表达量显著减少 ,与对照 VSMC相比差异显著 (P<0 .0 1 )。Ang (1 0 - 7mol/ L)刺激 2 4h后 ,与非转染 VSMCs相比 ,转染 VSMCs ATl m RNA明显减少 (P<0 .0 1 ) ,AT2m RNA明显增加 (P<0 .0 1 ) ;但两组间 PKC和 P38MAPK蛋白表达 ;3H- L eu及3H- Td R掺入量均无显著性差异 (P>0 .0 5 )。结论 :经 ATl A封闭后 ,能显著抑制 VSMC ATl m RNA表达 ,同时上调 AT2 m RNA。单纯封闭 ATlm RNA并不能有效阻断Ang 介导的 VSMCs蛋白核酸合成及 VSMCs生长相关的信号转导 ,

Aim:To evaluate the effect of AT 1 antisence nucleotide(AT 1A) on expression of subtypes of angiotensin Ⅱ(AngⅡ) receptor mRNA,PKC(protein kinase C),P 38 MAPK(mitogen activated protien kinase P 38 , and the syntheses of protein and nucleic acid in vascular smooth mnscle cells(VSMCs). Methods:AT 1 cDNA sequence (476bp) was cloned with RT PCR and inserted into PcDNA3.1(5.4kb) anti sense to construct an intact plasmid(PAT 1A).It was transfected into cultured VSMCs, and identified by RT PCR.Syntheses of protein and nucleic acid (by \{\} 3H Leucine and \{\} 3H thymidine incorporation), mRNA expressions of AT 1 and AT 2(by RT PCR),and protein expressions of P 38 MAPK and PKC(by western blot) between transfected and nontransfected VSMCs were compared after being stimulated with AngⅡl0 -7 mol/L for 24h. Results:We constructed PAT 1A successfully,RT PCR revealed AT 1 mRNA expressed less in trannsfected VSMCs than control( P <0.01). AT 1 mRNA expression was decreased( P <0.0l), AT 2 mRNA increased( P <0.0l),no apparent difference in\{\} 3H Leu and\{\} 3H TdR incorporation and expressions of PKC and P 38 MAPK( P >0.05) between transfected and control group after stimulation for 24 h by AngⅡ10 -7 mol/L. Conclusion:After being blocked by AT lA,expression of AT 1 mRNA in cultured VSMCs was markedly supressed,and AT 2 mRNA up regulated at the same time. It is shown that the syntheses of protein, nucleic acid, and growth related signaling protein in VSMCs could not be effectively interrupted by AT 1A blocking.