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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

  • 摘要: 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。

     

    Abstract: :Objective  To const ruct the expression vector containing CD :UPRT(cytosine deaminase :uracil phosphoribosylt ransferase) genes under the cont rol of the hTERT promoter and investigate it s specific killing effect s on human gast ric cancer cells SGC7901 in vi t ro. Methods  The hTERT promoter was PCR amplified and cloned into the p GL32Basic vector. The recombinant was t ransfected into SGC7901 cells and normal human fibroblast cells HL F to detect the t ranscriptional activities of the hTERT gene promot2 er. The expression vector containing CD :UPRT genes under the cont rol of the hTERT promoter named as hTERT2CD :UPRT was const ructed. This vector and the vector containing CD :UPRT genes under the cont rol of cytomegalovirus ( CMV) promoter named as pcDNA3. 12CD : UPRT were t ransfected into SGC7901 and HL F cells, respectively. The t ransfected cells were selected by G418. The expression of the CD gene was detected by RT2PCR and Western blot . MTT analysis was used to determine the cyto2 toxic effect s of the CD :UPRT/ 52FC system. Results  The hTERT promoter was PCR amplified success2 fully. Luciferase assay showed the relative luciferase activity of SGC7901 by the hTERT promoter was (21. 50 ±2. 15) % and that of HL F cells was only (0. 40 ±0. 07) %. The expression vector hTERT2CD : UPRT was successfully const ructed. Af ter stably t ransfected with pcDNA3. 12CD :UPRT, SGC7901 and HL F cells both expressed CD genes and were sensitive to 52FC, while positive only in SGC7901 cells af2 ter stably t ransfected with pcDNA 3. 12CD :UPRT. Conclusion  The hTERT promoter can specifically cont rol the CD :UPRT gene expression in SGC7901 cells but not in the normal cells and the CD :UPRT/52FC system under cont rol of the hTERT promoter can specially kill SGC7901 cells in vi t ro.

     

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