摘要
目的:利用多西环素开放(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(vascularsmoothmusclecells,VSMC),并经两次抗生素的筛选建立起了受四环素类似物多西环素(Doxycicline,Dox)紧密调控、表达血管紧张素Ⅱ2型受体(angiotensinⅡsubtype2receptors,AT2R)基因的双重稳定VSMC细胞系,在此基础上对细胞周期素激酶抑制蛋白-P21的表达受AngⅡ及其受体拮抗剂的影响进行研究。方法: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8组:①未转染对照组。②未转染+血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)组。③转染组。④转染+AngⅡ组。⑤转染+AngⅡ+Dox组。⑥转染+AngⅡ+Dox+CV-11974组。⑦转染+AngⅡ+Dox+PD123319组。⑧转染+AngⅡ+Dox+CV-11974+PD123319组。应用RT-PCR和免疫细胞化学染色技术,观察该双重稳定表达AT2R的VSMC细胞系中p21的mRNA及蛋白表达情况;采用AngⅡ及其1,2型其受体拮抗剂干预上述细胞,观测上述指标的变化。结果:加入Dox前转染组p21表达处于高水平,其mRNA及蛋白表达强度分别为118.363±19.225,0.196±0.013;AngⅡ1×10-7moL/L干预VSMC后p21mRNA及蛋白表达强度均显著降低,分别为51.761±4.429,0.032±0.014(t=13.10,
AIM:To introduce angiotensin Ⅱtype 2 receptors (AT2R) into the in vitro cultured vascular smooth muscle cells (VSMC) by using doxycycline (Dox) open conditional expression system of mammals, establish the double stable AT2R expression cell lines of VSMC, which was closely regulated by tetracycline analog Dox and expression AT2R, and investigate the effect of angiotensinⅡ(AngⅡ) and its receptor antagonist on the expression of cyclin dependent kinases inhibitor P21. METHODS:The Dox-regulatable double stable VSMC cell line to transfer AT2R gene was constructed with routine molecular biological methods, and the VSMC cell line was randomly divided into 8 groups: un-transfected control group, un-transfected+AngⅡgroup, transfected group, transfected+AngⅡgroup, transfected+AngⅡ+Dox group, transfected+AngII +Dox+CV-11974 (AT2R antagonist) group, transfected+AngII+Dox +PD123319 [angiotensin II type 1 receptors (AT1R) antagonist] group and transfected+AngII+Dox+CV-11974+PD123319 group.The mRNA and protein expressions of P21 in the VSMC cell line of the double stable expressed AT2R were observed with reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining respectively. The above cells were intervened by AngⅡ, AT1R antagonist (CV-11974) and AT2R antagonist (PD123319), and the changes of the aforementioned indexes were determined. RESULTS: Before Dox was added, there was high level of p21 expression in the transfected group, the expressions of its mRNA and protein were 118.363±19.225 and 0.196±0.013 respectively, which were significantly decreased after intervention of 1×10-7 moL/L AngⅡ(51.761 ±4.429, 0.032±0.014) (t=13.10,10.41, P< 0.01).After intervention of 1×10-7 moL/L AngII and add of Dox, P21 expressed highly, its mRNA and protein expressions were obviously higher than those in the transfected+AngII group (t=13.76,13.53,P< 0.01), and CV-11974 could further enhance the P21 expression, and its mRNA and protein expressions were obviously higher than those in the tr
出处
《中国临床康复》
CSCD
北大核心
2005年第7期42-44,i001,共4页
Chinese Journal of Clinical Rehabilitation
