摘要
目的 研究抗增殖性p21基因转染对体外培养的人血管平滑肌细胞(HVSMC)的增殖抑制作用及其作用机制。方法 通过腺病毒介导将外源性p21基因转入HVSMC,通过免疫印迹法(Western blot)检测p21基因的表达,原位末端标记法(TUNEL)、DNA片段梯度分析及流式细胞分析(FCM)观察靶细胞的细胞周期变化及凋亡,利用酶联免疫光度仪分析靶细胞的增殖抑制情况。结果 Western blot结果显示p21基因转染后的HVSMC可高水平表达p21蛋白,而另两组无表达。感染后5 d,Adp21组的细胞数为0.275 5±0.01(525 nm波长下吸光度A值),AdlacZ组为0.340 9±0.02,空白组为0.347 5±0.02,Adp21组较另两组的差异有统计学意义(P<0.01)。TUNEL、DNA片段梯度分析及FCM检测均提示靶细胞发生了凋亡。FCM检测还发现明显的细胞周期阻滞现象,Adp21组G0-G1期细胞比例为66.23％,S期细胞比例为10.18％,而另两组G0-G1期和S期细胞比例分别为:47.73％、30.56％和45.31％、32.34％。结论 复制缺陷性腺病毒在体外可有效介导外源性p21基因转染HVSMC,转染后的细胞除发生细胞周期阻滞外还出现明显的凋亡,其生长增殖受到抑制。
Purpose: To study the inhibition of HVSMC proliferation through anti-proliferation p21 gene transfection and its mechanism. Methods: An adenoviral expression vector with full length cDNA of p21 gene insert was cronstructed(Adp21) and transfected into HVSMC, the expression of exogenous p21 was tested by Western blot, the inhibition of target cells proliferation was investigated through the change of cell cycle and apoptosis which was detected by in situ nick-end labeling techniques(TUNEL), DNA ladder and flow cytometric(FCM) analysis. Results: Expression of p21 gene was confirmed by Western blot. Five days after transfected with Adp21, the cell number was 0. 275 5 ± 0.01 (absorbance at OD 525 nm), while 0.340 9 ± 0.02 in the AdlacZ group, and 0.347 5 ± 0.02 in the blank group. The proliferation of the p21 transfected HVSMC in vitro was significantly inhibited as compared with mock(AdlacZ) transfected HVSMC (P<0.01). Morphologically, the p21 transfected cells appeared apoptosis which was confirmed by TUNEL, DNA ladder and FCM analysis. FCM suggested cell cycle arrest occurred in p21 group, G0-G1 phase cells accounted for 66.23%, S phase cells only accounted for 10.18%. The other two groups were 47.73%, 30.56% and 45.31%, 32.34%. Conclusions: p21 gene mediated by replication-defective adenovirus can be transfected into HVSMC in vitro successfully. The transfected cells appear cell cycle arrest and apoptosis, and their proliferation are inhibited.
出处
《复旦学报（医学版）》
EI
CAS
CSCD
北大核心
2003年第4期367-370,375,共5页
Fudan University Journal of Medical Sciences
基金
上海市青年科技启明星计划基金(00QB14020)资助项目
