AT2R基因可调控表达对体外培养VSMC纤维黏连蛋白表达的影响

Effect of conditional expression of angiotensin Ⅱ type 2 receptor gene on mRNA and protein expression of fibronectin in rat vascular smooth muscle cells in vitro

目的利用Dox-on可调控哺乳动物表达系统,建立起了受四环素类似物Doxycycline(Dox)紧密调控、表达AT2R基因的双重稳定血管平滑肌细胞(vascular smooth muscle cells,VSMC),在此基础上对纤维黏连蛋白(fibronectin,FN)的表达受AngⅡ及其受体拮抗剂的影响进行研究。方法建立Dox可调控表达AT2R基因的双重稳定大鼠VSMC细胞,观察该VSMC细胞中AT2R受调控表达情况,以及血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)及其1型、2型受体拮抗剂干预上述细胞后FN的mRNA及蛋白表达情况变化。结果Dox-on可调控哺乳动物表达系统可成功介导AT2R基因在原代培养大鼠主动脉VSMC的表达,该表达受到Dox给予/去除的紧密调控;Dox干预可在48h内迅速诱导该VSMC细胞表达AT2R,AT2R表达在Dox干预后72h进一步增强(P<0.01)。AT2R基因的可调控表达抑制由于AngⅡ干预VSMC后引起FN表达的增强(P<0.01)。这一作用被AT1R拮抗剂CV-11974进一步增强(P<0.01);而被加入AT2R拮抗剂干预而取消;同时给予AT1R拮抗剂和AT2R拮抗剂时FN的表达与基础状态时的情况一致。结论AngⅡ干预增强FN的表达,该作用是通过AT1R介导的;经Dox诱导表达AT2R基因可以明显抑制这一生物学作用,说明在这一生物学效应上,AT2R具有与AT1R相拮抗的生物学功能。

Objective To investigate the effect of Ang Ⅱ and its receptor antagonist on the fibronectin （FN） mRNA and protein expression in VSMC of double stable AT2R expression. Methods VSMC was established, in which the expression levels of AT2R is tightly controlled by Doxycycline （Dox）. The VSMC were treated with Ang Ⅱ , Dox, CV-11974, PD123319. The effect of AT2R overexpression on the mRNA and protein expression of FN in VSMC were detected and the effects of AT1R antagonist （CV-11974） and AT2R antagonist AT2R expression were induced obviously in double stable VSMC within 48 h by Dox and further enhanced after 72 h （P 〈 0.01 ）. In the well established double stable VSMC, the addition of Ang Ⅱ resulted in an increase of the FN expression （P 〈0.01 ）. The FN expression decreased after Dox treatment （P 〈0.01 ）. The decreased expression of FN induced by Dox was further enhanced by CV-11974 （ P 〈 0.01 ）, and were removed by PD123319. There was no difference in FN expression between CV-11974 and PD123319 simultaneous group and control group. Conclusion The enhancement effect of Ang Ⅱ on the FN expression is mediated by AT1Rand inhibited by Dox-induced AT2R expression. It is suggested that AT2R acts an antagonistic effect against AT1 R in those biological effect.