人端粒酶逆转录酶启动子调控下胞嘧啶脱氨酶胸苷激酶融合基因治疗卵巢癌的体外研究  

In vitro treatment of ovarian cancer cells with cytosine deaminase-thymidine kinase fusion disuicide gene therapy system under the control of human telomerase reverse transcriptase gene promoter

作　　者：孔北华[1] 宋悦[1] 马道新[2] 曲迅[1] 江森[1] 

机构地区：[1]山东大学齐鲁医院妇产科,济南250012 [2]山东大学齐鲁医院中心血液病研究室,济南250012

出　　处：《中华妇产科杂志》2004年第6期390-395,共6页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金资助项目 ( 3 0 2 713 61)

摘　　要：目的　探讨人端粒酶逆转录酶 (hTERT)启动子调控下的融合双自杀基因———胞嘧啶脱氨酶 胸苷激酶 / 5 氟胞嘧啶 +更昔洛韦 (CD TK/ 5 FC +GCV)系统对人卵巢癌细胞及正常细胞的体外杀伤作用。方法　应用脂质体转染法将hTERT核心启动子报告质粒pBTdel 2 79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。应用基因克隆技术分别构建hTERT启动子和巨细胞病毒 (CMV)启动子调控下的CD TK基因真核表达载体pBTdel 2 79 CD TK和pcDNA3 CD TK ,以及hTERT启动子调控下的TK基因表达载体pBTdel 2 79 TK。用四甲基偶氮唑蓝 (MTT)法和流式细胞术检测pBTdel 2 79 CD TK/ 5 FC +GCV系统和pcDNA3 CD TK/ 5 FC +GCV系统对上述 3种细胞不同的杀伤作用 ;应用RT PCR技术检测pBTdel 2 79 CD TK和pcDNA3 CD TK转染前后 3AO和NOEC细胞中CD和TK基因的表达情况。用扫描电子显微镜观察pBTdel 2 79 CD TK/ 5 FC +GCV作用后 3AO细胞的形态变化。结果　卵巢癌细胞系 3AO中hTERT启动子活性增高 ,为 2 4 1% ,RT PCR检测hTERTmRNA为阳性 ,而在正常细胞NOEC和HELF中 ,hTERT启动子活性仅为 0 3%和 0 7% ,hTERTmRNA为阴性。Objective To investigate the in vitro effects of cytosine deaminase-thymidine kinase/ 5-fluorocytosine+ ganciclovir(CD-TK/5-FC+GCV) on ovarian cancer cells and normal cells using a human telomerase reverse transcriptase (hTERT) promoter-driven vector system. Methods hTERT promoter activity was determined by luciferase assay after the plasmids of pBTdel-279 containing hTERT core promoter were transfected into ovarian carcinoma-derived cell line 3AO, human normal ovarian epithelial cell (NOEC) and human embryonic lung fibroblast (HELF). hTERT mRNA expression levels of these cells were determined by reverse transcription polymerase chain reaction (RT-PCR). Eukaryotic expression vectors containing CD-TK fusion disuicide genes under the control of hTERT promoter (pBTdel-279-CD-TK) and cytomegalovirus(CMV) promoter (pcDNA3-CD-TK), as well as vectors containing TK gene under hTERT promoter control (pBTdel-279-TK), were constructed and transfected into 3AO, NOEC and HELF cells by cationic liposome. Following the transfection with CD and TK,5-FC and GCV of different doses were added and methyl thiazolyl tetrazolium(MTT) and flow cytometry methods were applied to investigate the antitumor effects of pBTdel-279-CD-TK/5-FC+GCV and pcDNA3-CD-TK/ 5-FC+GCV systems. pBTdel-279-TK/GCV was considered as a control. RT-PCR was used to detect CD and TK genes in ovarian cancer cells and normal cells before and after the transfection of pBTdel-279-CD-TK or pcDNA3-CD-TK. Scanning electron microscopy (SEM) was used to examine the morphology of 3AO after the action of pBTdel-279-CD-TK/ 5-FC+GCV. Results hTERT promoter activity was 24.1% and hTERT mRNA expression was positive in ovarian cancer cell 3AO, whereas in NOEC or HELF the activity was 0.3% or 0.7% and hTERT mRNA expression was negative. Expression vectors of pBTdel-279-CD-TK, pcDNA3-CD-TK and pBTdel-279-TK were successfully constructed. Compared with the effects of pBTdel-279-TK/GCV, the pBTdel-279-CD-TK/5-FC+GCV system was more efficient on hTERT positive ovarian cancer cel

关 键 词：人端粒酶逆转录酶 启动子 调控 胞嘧啶脱氨酶 胸苷激酶 基因治疗 卵巢癌 体外研究 

分 类 号：R737.31[医药卫生—肿瘤]