hTERT启动子调控的融合自杀基因CD:UPRT载体的构建及其应用

Construction of Cytosine Deaminase:Uracil Phosphoribosyltransferase/5-fluorocytosine Gene Therapy System under Control of Human Telomerase Reverse Transcriptase Promoter and Its Application

目的构建hTERT启动子调控的融合自杀基因CD:UPRT表达载体,研究其对人胃癌细胞SGC7901的体外靶向性杀伤作用。方法PCR扩增hTERT核心启动子片段,克隆入荧光素酶报告基因质粒pGL3-Basic,检测hTERT启动子在人胃癌细胞SGC7901和人成纤维细胞HLF中的转录活性。构建hTERT启动子调控的CD:UPRT基因表达载体hTERT-CD:UPRT,将其和CMV启动子调控的CD:UPRT基因表达载体pcDNA3.1-CD:UPRT用脂质体转染法分别转染入SGC7901和HLF细胞,筛选稳定表达细胞系,用RT-PCR和Western blot方法检测CD基因的表达,用MTT法检测5-FC对转染细胞的杀伤作用。结果成功克隆hTERT核心启动子;荧光素酶活性检测显示,hTERT启动子在SGC7901细胞中的转录活性为阳性对照的(21.50±2.15)%,而在HLF细胞中仅有背景活性。成功构建hTERT启动子调控的CD:UPRT基因表达载体,转染pcDNA3.1-CD:UPRT的SGC7901和HLF细胞以及转染hTERT-CD:UPRT的SGC7901细胞在mRNA和蛋白质水平均可检测到CD基因的表达,且对5-FC敏感;而转染hTERT-CD:UPRT的HLF细胞未检测到CD基因的表达,对5-FC不敏感。结论构建的hTERT启动子调控的融合自杀基因系统CD:UPRT/5-FC能在体外靶向性杀伤SGC7901细胞。

Objective To construct the expression vector containing CD：UPRT（cytosine deaminase： uracil phosphoribosyhransferase） genes under the control of the hTERT promoter and investigate its specific killing effects on human gastric cancer cells SGC7901 in vitro. Methods The hTERT promoter was PCR amplified and cloned into the pGL3-Basic vector. The recombinant was transfected into SGC7901 cells and normal human fibroblast cells HLF to detect the transcriptional activities of the hTERT gene promoter. The expression vector containing CD： UPRT genes under the control of the hTERT promoter named as hTERT-CDtUPRT was constructed. This vector and the vector containing CD..UPRT genes under the control of cytomegalovirus （CMV） promoter named as pcDNA3. 1-CD.. UPRT were transfected into SGC7901 and HLF cells, respectively. The transfected cells were selected by G418. The expression of the CD gene was detected by RT-PCR and Western blot. MTT analysis was used to determine the cytotoxic effects of the CD： UPRT/5-FC system. Results The hTERT promoter was PCR amplified successfully. Luciferase assay showed the relative luciferase activity of SGC7901 by the hTERT promoter was （21.50 ± 2. 15） % and that of HLF cells was only （0. 40 ± 0.07） %. The expression vector hTERT-CD： UPRT was successfully constructed. After stably transfected with pcDNA3. 1-CD.UPRT, SGC7901 and HLF cells both expressed CD genes and were sensitive to 5-FC, while positive only in SGC7901 cells after stably transfected with pcDNA 3.1-CD： UPRT. Conclusion The hTERT promoter can specifically control the CD： UPRT gene expression in SGC7901 cells but not in the normal cells and the CD： UPRT/ 5-FC system under control of the hTERT promoter can specially kill SGC7901 cells in vitro.