摘要
目的:探讨血管平滑肌细胞(vascularsmoothmusclecell,VSMC)转染表达血管紧张素II(angiotensinII,AngII)2型受体(angiotensinIItype2receptor,AT2R)后其1型受体(angiotensinIItype1receptor,AT1R)表达所受的影响。方法:用同源重组方法构建带AT2R基因的重组复制缺陷型腺病毒载体(AdCMV-AT2R),体外转染VSMC,分别用流式细胞仪检测AT1R、AT2R细胞转染表达率、用免疫组织化学法和免疫荧光法检测其膜表达、以反转录聚合酶链式反应法(RT-PCR)和蛋白印迹法检测其mRNA和蛋白表达。结果:AdCMV-AT2R转染后,随着转染表达时间延长,AT2R细胞表达率呈显著增加趋势,48h最高表达率达89.51%,AngII作用与否及不同浓度AngII作用对AT2R表达无显著影响。而转染前后AT1R表达相对较稳定,受不同浓度AngII作用,其表达明显呈增加趋势。AT2R峰值表达时,免疫组织化学法和免疫荧光法检测其膜表达结果也提示AT2R表达随转染表达时间延长显著增加,转染前后AT1R表达无明显变化,在一定浓度范围内,AngII刺激对AT2R表达无显著影响,却显著增加AT1R的膜表达。RT-PCR法和蛋白印迹法检测AT1R和AT2R的mRNA和蛋白表达结果与其细胞表达率和膜表达的检测结果相一致。结论:AT2R转染表达并发挥其生物学作用时,对AT1R的表达无明显影响,VSMC转染表达AT2R后,
AIM: To explore the expression of angiotensin II (AngII) type 2 receptor (AT2R) and its effect on the expression of angiotensin II (AngII) type 1 receptor (AT1R) in vascular smooth muscle cell (VSMC). METHODS: The recombinant adenoviral vector, AdCMV AT2R, containing rat AT2R gene was constructed by homologous recombination, and then it was used to transfer AT2 receptor gene to rat VSMC in vitro. The expression of AT2R, AT1R mRNA was detected by RT PCR and the rate of expression in VSMC was determined by flow cytometer, the protein and mRNA of AT2R and AT1R in membrane of VSMC was detected by immunohistochemistry and immunofluorescence respectively. RESULTS: The expression rate of AT2R in VSMC was increased significantly after transferred by AdCMV AT2R with the time in transferred VSMC, and the peak value detected by flow cytometry was about 89.51% at 48 h. The results of RT PCR, immunohistochemistry and immunofluorescence showed that the mRNA transcription and protein translation of AT2R were increased significantly. The longer VSMCs were transfected by AdCMV AT2R, the more mRNA and protein of AT2R were detected. There were no significant changes of AT1R expression to be found during AT2R expression. The stimulant of different consistence AngII had no significant effect on the expression of AT2R, but increased the expression of AT1R significantly and showed concentration- dependent manner. CONCLUSION: The expression of AT2R can not affect the expression and of AT1R in VSMC before the bio function. Dependent on the concentration, AngII can increase the expression of AT1R after the expression of AT2R. Therefore, AT2R transfection is goof for the regulation of VSMC function.
出处
《中国临床康复》
CSCD
2004年第6期1054-1056,共3页
Chinese Journal of Clinical Rehabilitation
