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[目的]构建人端粒酶逆转录酶启动子(hTERTp)调控下的自杀基因—胞嘧啶脱氨酶-胸苷激酶/5-氟胞嘧啶(CD-TK/5-FC)系统对人卵巢细胞及正常细胞株的体外杀伤作用。[方法]应用脂质体转染法将hTERT核心启动子启动质粒pBTdel-279转染人卵巢癌细胞系3AO、正常卵巢上皮细胞(NOEC)和人胚肺成纤维细胞株HELF,用荧光素酶分析检测hTERT启动子活性;应用RT-PCR技术检测hTERTmRNA的表达水平;用四甲基偶氮唑蓝(MTT)法及流式细胞仪检测pBTdel-279-CD-TK/5-FC系统和pcDNA3-CD-TK/5-FC系统对上述3种细胞不同的杀伤作用;利用RT-PCR技术检测转染前后3AO和NOEC细胞中CD和TK基因的不表达情况。[结果]卵巢癌细胞系3AO中hTERT启动子活性增高,为23.2%,与pBTdel-279-TK/GCV系统作用相比,pBTdel-279-CD-TK/5-FC+GCV系统对端粒酶阳性的卵巢癌细胞系3AO的杀伤作用显著增强,对于端粒酶阴性正常细胞NOEC和HELF则无杀伤作用;pcDNA3-CD-TK转染后,3种细胞中均可检测到CD和TK基因;pBTdel-279-TK转染后,3AO可检测到CD和TK基因,正常细胞中则呈阴性。[结论]人端粒酶逆转录酶启动子调控下的CD自杀基因对卵巢癌细胞株选择性杀伤作用治疗是一种高效、靶向的卵巢癌基因治疗方式。
[Objective] To construct a human CD gene in human ovary by using the CD86 / 5-FC system, a suicide gene regulated by human telomerase reverse transcriptase promoter (hTERTp) In vitro cytotoxicity of cells and normal cell lines. [Method] The hTERT core promoter promoter plasmid pBTdel-279 was transfected into human ovarian cancer cell line 3AO, normal ovarian epithelial cells (NOEC) and human embryonic lung fibroblast cell line HELF by liposome transfection method. The luciferase The expression of hTERT mRNA was detected by RT-PCR. The expression of pBTdel-279-CD-TK / 5-FC and pcDNA3 were detected by MTT assay and flow cytometry -CD-TK / 5-FC system. The RT-PCR was used to detect the non-expression of CD and TK genes in 3AO and NOEC cells before and after transfection. [Result] The hTERT promoter activity in ovarian cancer cell line 3AO increased by 23.2%. Compared with the effect of pBTdel-279-TK / GCV system, pBTdel-279-CD-TK / 5- The positive ovarian cancer cell line 3AO significantly enhanced the killing effect on telomerase negative normal cells NOEC and HELF no killing effect; pcDNA3-CD-TK transfected cells were detected in all three CD and TK genes; After transfection with pBTdel-279-TK, the CD3 and TK genes were detected in 3AO and negative in normal cells. [Conclusion] CD suicide gene under the control of human telomerase reverse transcriptase promoter is a highly effective and targeted ovarian cancer gene therapy for the selective killing of ovarian cancer cell lines.