热诱导肿瘤特异性基因治疗载体的靶向性鉴定
Targeting validation of a heat-induced, tumor-specific gene therapy vector

摘要：目的 检测已构建热反应元件修饰的端粒酶逆转录酶hTERT基因启动子(8HSEs-hTERTp)调控的基因表达载体肿瘤特异性和热诱导特性。方法 ①RT-PCR、Western blot和免疫细胞化学检测细胞内hTERT mRNA和蛋白表达水平，选择hTERT高表达和低表达的细胞株作为该研究阳性细胞株和阴性细胞株；②收集前期已包装成功的热反应元件8HSEs和人端粒酶逆转录酶启动子hTERTp联合调控自杀基因PNP表达的慢病毒表达载体8HhP病毒液，感染hTERT+ SW480细胞及hTERT－ MKN28细胞、hTERT－ MRC-5细胞，细胞免疫荧光技术检测该治疗载体的肿瘤特异性及热反应特性；并用Western blot和RT-PCR分别检测热刺激条件下目的基因PNP在不同hTERT水平细胞株中的表达水平。结果 hTERT mRNA及蛋白在SW480细胞中高表达，在MKN28细胞和MRC-5细胞中低表达甚至阴性表达；免疫荧光、RT-PCR和Western blot结果显示，8HSEs-hTERTp调控的目的基因PNP只有在43℃处理下的SW480细胞中呈现高表达，而在阴性细胞株MKN28和MRC-5中PNP表达较少，而常温下SW480细胞中PNP的表达水平明显低于热处理组。结论 8HSEs修饰的hTERT启动子可以明显提高治疗载体的肿瘤特异性及热反应特性。
ABSTRACT: Objective To verify tumor- and heat-specificity to hyperthermia stimulation of a gene therapy vector containing the gene PNP regulated by the eight heat shock elements (8HSEs) and the hTERT promoter. Methods ① The expression of hTERT was measured by RT-PCR, Western blot and immunocytochemistry assays. ②The recombinant lentiviral vector pLVX-8HSEs-hTERTp-PNP-3FLAG(8HhP) containing an hTERT promoter and 8 copies of the HSEs, which was constructed in our previous study, was collected. 8HhP was transfected into hTERT+ SW480 cells, hTERT－ MKN28 cells and hTERT－ MRC-5 cells; then the tumor- and heat-specificity was verified by immunofluorescence. Western blot and RT-PCR were used to examine the expression of PNP under heated conditions. Results hTERT mRNA and protein expressions were high in SW480 cells, but low or negative in MKN28 and MRC-5 cells. The expressions of PNP protein and mRNA in 8HhP/SW480 cells were obviously increased only under heated conditions, which was confirmed by Western blot, RT-PCR and immunofluorescence. In contrast, the expressions of PNP protein and mRNA in 8HhP/MKN28 and 8HhP/MRC-5 cells were low or negative even under heated conditions. Conclusion The hTERT promoter modified by 8HSEs can increase the gene therapy vector specificity to hyperthermia and tumors