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目的 寻求肿瘤反义基因治疗的新途径。方法 应用分子克隆技术构建增殖细胞核抗原 (PCNA)基因反义真核表达载体 ,转染膀胱癌EJ细胞 ,通过免疫荧光、逆转录 聚合酶链反应(RT PCR)、噻唑蓝 (MTT)比色法、克隆形成实验动态检测转染 1～ 7d后癌细胞PCNA基因表达和体外增殖活性变化。结果 所获反义表达载体 pLAPSN转染可使癌细胞PCNA蛋白、mRNA表达水平分别抑制 16.74%～ 84.2 1% (P <0 .0 5 )、2 3.2 7%～ 86.15 % (P <0 .0 5 ) ,增殖活性抑制2 7.91%～ 62 .0 7% (P <0 .0 1) ,克隆形成能力降低 5 0 .81% (P <0 .0 1)。结论 应用反义RNA技术阻断PCNA基因的表达 ,能有效抑制癌细胞的体外增殖活性 ,是膀胱癌基因治疗的合理策略之一。
Objective To search for new ways of antisense gene therapy in cancer. Methods The antisense eukaryotic expression vector of proliferating cell nuclear antigen (PCNA) gene was constructed by molecular cloning technique and transfected into EJ cells of bladder cancer. The expression of PCNA gene was detected by immunofluorescence, reverse transcription polymerase chain reaction (RT PCR), MTT assay The clonogenic assay was used to detect the expression of PCNA gene and the proliferative activity in vitro after transfection for 1-7 days. Results The antisense expression vector pLAPSN could inhibit the expression of PCNA protein and mRNA in cancer cells from 16.74% to 84.2% (P <0.05), from 3.227% to 86.15% (P <0. 0) 5). The proliferative activity was inhibited by 7.91% -62.07% (P <0.01) and the clonogenic capacity was decreased by 50.81% (P <0.01). Conclusion Antisense RNA technology can block the expression of PCNA gene and effectively inhibit the proliferation of cancer cells in vitro. It is one of the reasonable strategies for gene therapy of bladder cancer.