摘要
为探讨腺病毒介导反义人AT1R转染对培养的人血管平滑肌细胞 (VSMCs)增殖和凋亡的影响 ,用重组反义人AT1R的复制缺陷型腺病毒 (Ad CMV ahAT1)转染体外培养的人VSMCs,用RT PCR和免疫组化法检测AT1R的表达 ,用流式细胞仪检测VSMCs的增殖指数和凋亡率 ,绘制DNA合成直方图。结果显示 :与对照组比较 ,转染Ad CMV ahAT1后 4 8h的VSMCs,AT1RmRNA低 5 0 % ,AT1R蛋白呈低表达 (P <0 0 1) ;给予AngⅡ刺激的VSMCs的增殖指数明显增加 (与DMEM组比 ,P <0 0 1) ,转染Ad CMV ahAT1组的增殖指数则显著降低 (与DMEM组比P <0 0 5 ,与AngⅡ对照组比P <0 0 1)。Ad CMV ahAT1组在二倍体峰之前出现凋亡峰 ,各时相点细胞凋亡率分别为 18%(6h)、2 0 % (12h)、2 8% (2 4h)、2 6 % (4 8h)、3% (72h)和 1% (96h) ,而在其它各对照组未出现凋亡峰。这些提示腺病毒介导的反义人AT1R转染 ,通过抑制AT1R的表达 ,明显抑制人VSMCs的增殖和AngⅡ刺激的人VSMCs增殖 ,并诱导VSMCs凋亡。
To identify the role of human angiotensin Ⅱ (AngⅡ) type 1 receptor (AT1R) antisense cDNA (ahAT1) on the proliferation and apoptosis of cultured human artery tissue smooth muscle cells (VSMCs) induced by AngⅡ, Two recombinant adenoviral vectors, Ad/CMV.ahAT1 containing full length antisense cDNA targeting to human AT1R mRNA, and Ad/CMV.LacZ containing LacZ (report gene) were constructed by orientation clone technique and homologous recombination, and then transfected into VSMCs from human umbilical artery. After stimulation for 24h by AngⅡ 10 -7 mol/L, AT1R expression was detected by RT-PCR and immunohistochemical method, Proliferation index (PI) and apoptosis of VSMCs were determined by flow cytometry. Ad/CMV.LacZ transfected and nontransfected VSMCs served as control. Forty eight hours after being transfected Ad/CMV.ahAT1 into VSMCs, the level of AT1R mRNA decreased markedly (50% of control-group) and AT1R protein expression was significantly less than that in other groups (P<0.01 compare with control-group and Ad/CMV.LacZ-group respectively). AngⅡ markedly increased PI of VSMCs in a time-dependent manner (P<0.05 or P<0.01 compare with control). The increased PI of VSMCs was blocked to control level by transfecting Ad/CMV.ahAT1 into VSMCs named as Group AngⅡ-Ad/CMV.ahAT1 (P<0.05 compare with control, <0.01 compare with AngⅡ stimulated only). But there was no statistic difference in these parameters between Group AngⅡ-Ad/CMV.LacZ and Group AngⅡ. Apoptosis peak emerged only in Group AngⅡ-Ad/CMV.ahAT1. The rate of apoptosis in those VSMCs used Ad/CMV.ahAT1 and AngⅡ was 1% to 28%. These results indicated that antisense cDNA targeted to human AT1R transfer in vitro mediated by adenoviral vector had a powerful inhibitory effect on AngⅡ-induced proliferation of VSMCs by attenuating AT1R expression.
出处
《基础医学与临床》
CSCD
北大核心
2004年第4期408-413,共6页
Basic and Clinical Medicine
基金
湖北省自然科学基金 (2 0 0 1ABB16 7)
卫生厅项目 (WJ0 15 80 )
