PCNA反义cDNA基因转导抑制人膀胱癌细胞增殖的研究

A study of antisense cDNA targeting proliferation cell nuclear antigen gene on the inhibition of human bladder cancer cells

目的 探讨增殖细胞核抗原 (PCNA)基因反义cDNA对人膀胱癌细胞体外增殖活性的抑制作用。 方法 构建PCNA反义cDNA真核表达载体并转染膀胱癌EJ细胞 ,通过细胞生长曲线、MTT比色分析、H3 TdR掺入法检测癌细胞增殖活性 ,流式细胞仪 (FCM )分析细胞周期时相变化 ,SABC免疫组化观察癌细胞PCNA蛋白表达水平。 结果 PCNA反义cDNA导入后 ,膀胱癌EJ细胞生长速率显著减慢 (P <0 .0 1) ,增殖活性抑制率 5 9.0 2 % (P <0 .0 5 ) ,DNA合成速率降低 5 2 .31% (P <0 .0 1) ,S期细胞减少 ,细胞周期阻滞于G0 /G1期 ,PCNA蛋白表达显著减弱 (P <0 .0 5 ) ,差别均有显著性意义。 结论 PCNA反义cDNA基因转导能有效抑制膀胱癌细胞的PCNA蛋白表达及体外增殖活性 。

Objective To study antisense cDNA targeting proliferation cell nuclear antigen (PCNA) gene on the inhibition of human bladder cancer cells in vitro. Methods The antisense eukaryotic expression vector of human PCNA cDNA was constructed by molecular cloning techniques, which transfected the bladder cancer EJ cells with Lipofectamine 2000. The proliferation of cancer cells was detected by cell growth curves, MTT and H 3 TdR assays. The changes of cell cycle phases were studied by flowcytometry (FCM). The expression of PCNA was observed by SABC immunohistochemical method. Results After the antisense PCNA cDNA being transferred , the growth of bladder cancer EJ cells was decreased significantly ( P <0.01) with the proliferation being inhibited by 59.02%( P <0.05).The DNA synthesis rates were blocked by 52.31%( P <0.01). Cell cycle analysis showed there was a decrease in S phase and an increase in G0/G1 phase. The protein expression of PCNA was inhibited significantly ( P <0.05). Conclusions Gene transduction of antisense cDNA targeting PCNA could inhibit in vitro the proliferation of bladder cancer cells through blocking the protein expression of PCNA . This provided an effective way for gene therapy of cancer.