条件表达AT2R基因对体外培养血管平滑肌细胞骨桥蛋白mRNA表达的影响

Effect of conditional expression of angiotensin Ⅱ type 2 receptor gene on mRNA expression of osteopontin in rat vascular smooth muscle cells in culture

目的利用Dox-on可调控哺乳动物表达系统将AT2R基因引入体外培养的血管平滑肌细胞(vascular smooth muscle cells,VSMC),并经2次抗生素的筛选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VSMC细胞系,在此基础上对骨桥蛋白(osteopontin,OPN)的mRNA表达受AngⅡ及其受体拮抗剂的影响进行研究。了解其在再狭窄防治中的意义。方法本研究通过常规分子生物学方法,建立Dox可调控表达AT2R基因的双重稳定大鼠VSMC细胞系。将该VSMC细胞系随机分为未转染对照组、转染组及AngⅡ、Dox、CV-11974、PD123319分别或同时干预组共8组。应用免疫印迹方法、RT-PCR技术,观察该VSMC细胞系中AT2R受调控表达情况,以及血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)及其1型、2型其受体拮抗剂干预上述细胞后OPN的mRNA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细胞系表达AT2R,AT2R表达在Dox干预后72h进一步增强,差异有统计学意义(P<0.01)。AT2R基因的可调控表达抑制由于AngⅡ干预VSMC后引起的OPN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OPN的表达与基础状态时的情况一致。结论AngⅡ干预增强OPN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。AT2R基因经诱导后可以通过调节OPN表达,进而可能对VSMC迁移、增殖和细胞外基质形成进行有效调控。

Objective To investigate the effect of AngⅡ and its receptor antagonist on the osteopontin （OPN） mRNA expression in double stable AT2R expression cell lines of VSMC. Methods To construct the tetracycline-regulatable vector system to transfer angiotensin Ⅱ （Ang Ⅱ ） type 2 receptor （AT2R） gene by homologous recombination, and establish the double stable cell lines of rat vascular smooth muscle cells（VSMC） to express AT2R in two step of selection by antibiotic in which the expression levels of AT2R is tightly controled by doxycycline（Dox）. The VSMC were divided into 8 groups randomly： un-transfected control group,transfected group, and treatment with AngⅡ ,Dox,CV-11974,PD123319 apart or simultaneously group. The effect of over-expression of AT2R on the mRNA expression of OPN were detected by RT-PCR and mmunohistochemistry respectively. And the effects of AT1R antagonist （CV-11974） and AT2R antagonist （PD123319） on aforementioned target were studied. Results The doxcycline（Dox） - on gene expression system can generate high level and regulable expression of AT2R which tightly controled by addition/removal Dox. The expression of AT2R were induced obviously in double stable VSMC within 48h by addition Dox and further . enhanced after 72h （P〈0. 01）. In the well established double stable VSMC lines, addition of Ang Ⅱ resulted in a increase of the expression of the OPN （P〈0.01）. The OPN expression decreased by treatment with Dox（P〈0.01）. Dox-induced decreased expression of OPN were further enhanced by CVq1974（P〈0. 01）, and were removed by PD123319. There are no differ ence in expression of OPN between CV-11974 ,PD123319 simultaneously group with control group. Conclusion The enhancement effect of AngⅡ on the expression of OPN is mediated by AT1R and inhibited by AT2R expression. It is suggestded that AT2R acts an antagonistic effect against AT1R in those biological effect. The regulable expression of AT2R might be control of vascular smooth muscle cell proliferation, migration and phenotype by regulation the the expression of OPN.