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目的:探讨血管紧张素Ⅱ2型受体(AT2R)基因体外及体内转染对血管平滑肌细胞(VSMC)增殖核抗原(PCNA)表达的影响。方法:细胞爬片及大鼠颈动脉球囊损伤后,局部转染带有绿色荧光蛋白报告基因的AT2R重组腺病毒载体(pAd-AT2R)或病毒载体(pAd-GFP),于细胞转染后48h及术后14d取材,应用免疫组织化学染色、免疫荧光双标染色和激光共聚焦技术检测了VSMC及血管中AT2R与PCNA的表达关系。结果:pAd-AT2R转染VSMC后,在激光共聚焦显微镜下胞质及胞核有绿色荧光蛋白表达;同时在胞质有红色荧光AT2R表达,胞核无蓝色荧光,PCNA表达阴性,无红色荧光AT2R表达的VSMC胞核有蓝色荧光,PCNA表达阳性。pAd-AT2R在体转染大鼠颈动脉后,新生内膜及中膜中PCNA阳性表达率显著低于未转染组和pAd-GFP组[(27.29±5.81)%∶(72.25±4.47)%,(68.43±9.12)%,P<0.01];在激光共聚焦显微镜下,绿色荧光及红色荧光AT2R主要分布于新生内膜、中膜、外膜,内膜散在蓝色荧光PCNA表达,在红、绿色荧光处无蓝色荧光,PCNA表达阴性,无红、绿色荧光处,有蓝色荧光,PCNA表达阳性。结论:AT2R基因体外及体内转染可抑制VSMCPCNA表达,ATR基因转染能抑制新生内膜增生。
Objective: To investigate the effect of AT2R gene transfection on the expression of proliferating cell nuclear antigen (PCNA) in vitro and in vivo of vascular smooth muscle cells (VSMCs). METHODS: The AT2R recombinant adenoviral vector (pAd-AT2R) or the viral vector (pAd-GFP) carrying the green fluorescent protein reporter gene was transfected locally after the cell slide and carotid artery balloon injury in rats. After transfection 48h and 14d postoperatively. The expression of AT2R and PCNA in VSMCs and blood vessels were detected by immunohistochemistry, immunofluorescence double staining and confocal laser scanning microscope. Results: The expression of green fluorescent protein in the cytoplasm and nucleus of pAd-AT2R transfected VSMCs was observed under laser scanning confocal microscope. At the same time, there was a red fluorescence AT2R expression in the cytoplasm, the nuclei were blue fluorescence, PCNA expression was negative, no red Fluorescence AT2R expression of VSMC nuclei blue fluorescence, PCNA expression was positive. The positive rate of PCNA in neointima and media of pAd-AT2R transfected rat carotid artery was significantly lower than that of untransfected group and pAd-GFP group [(27.29 ± 5.81)%] (72.25 ± 4.47% , (68.43 ± 9.12)%, P <0.01]. Under confocal laser scanning microscope, green fluorescence and red fluorescence AT2R were mainly distributed in neointimal, medial and adventitia, , No blue fluorescence at green fluorescence, negative PCNA expression, no red or green fluorescence, blue fluorescence and positive PCNA expression. CONCLUSION: Transfection of AT2R gene inhibits the expression of VSMCCPCNA in vitro and in vivo. ATR gene transfection can inhibit neointimal hyperplasia.