论文部分内容阅读
理想的基因治疗要求实现目的基因的可控性表达。一种应用热能诱导激活目的基因表达,以达到靶向治疗目的的新方法已经问世。 将绿色荧光蛋白(GFP)基因、人TNF-α基因、鼠IL-12(mIL-12)基因分别克隆人质粒p△EsplA,使其处于400 bp热休克蛋白(hsp)启动子控制之下。将质粒转染包装细胞,产生重组腺病毒,分别命名为AdhspGFP,AdhspTNF-α和AdhspmIL-12。将其分别转染肿瘤靶细胞,短时间热处理后,应用荧光活化细胞分析(FAGS),荧光显微镜、Western杂交分析及ELISA方法检测目的基因表达产物。进行肿瘤生长抑制试验,检测热能对基因治疗后肿瘤生长的抑制效果。 结果:①体外热诱导基因表达的温度反应和动力学变化:鼠4T1乳腺癌细胞转染AdhspGFP后,分别以39～44℃加温20 min。24小时后检测显示GFP启动子活化开始于
Ideal gene therapy requires the controlled expression of the gene of interest. A new approach to the use of thermal energy to induce the activation of target gene expression for targeted therapy has emerged. The green fluorescent protein (GFP) gene, human TNF-α gene, and murine IL-12 (mIL-12) gene were respectively cloned into the human plasmid pΔEsplA to be under the control of the 400 bp heat shock protein (hsp) promoter. The plasmids were transfected into packaging cells to produce recombinant adenoviruses named AdhspGFP, AdhspTNF-α and AdhspmIL-12, respectively. They were transfected with target tumor cells and heat-treated cells were analyzed by fluorescence activated cell analysis (FAGS). Fluorescence microscopy, Western blot analysis and ELISA were used to detect the target gene expression products. A tumor growth inhibition test was performed to examine the inhibitory effect of heat on tumor growth after gene therapy. RESULTS: 1The temperature response and kinetics of heat-induced gene expression in vitro: After transfected with AdhspGFP, 4T1 breast cancer cells were heated at 39-44°C for 20 min. Detection after 24 hours showed that GFP promoter activation started at