背景与目的:有学者用人端粒酶逆转录酶(TERT)启动子替换野生型腺病毒E1A自身的启动子,构建了一种肿瘤特异性增殖病毒,即溶瘤腺病毒Ad.TERT。细胞实验和动物实验表明,Ad.TERT有很好的抗癌作用。本研究目的是要在Ad.TERT中插入凋亡基因trail生成Ad.TERT-TRAIL,观察其是否具有更强的杀伤肿瘤细胞的功效并研究其机理。方法:通过腺病毒包装质粒pBH GE3与携带trail的小质粒pZhTERT-trail在H EK293细胞中同源重组构建Ad.TERT-TRAIL病毒,然后用PCR鉴定,并用W estern blot检测E1A、trail基因的表达以进一步确证。用结晶紫染色法和M TT法检测Ad.TERT和Ad.TERT-TRAIL对肿瘤细胞和正常细胞杀伤作用的差异。用W estern blot检测Caspase-3表达量以观察肿瘤细胞的凋亡情况,同时用流式细胞仪测定肿瘤细胞的凋亡率。结果:PCR扩增出来的条带为860bp,即为trail基因。W estern blot结果显示,Ad.TERT-TRAIL中的E1A和trail只在肿瘤细胞中表达,说明Ad.TERT-TRAIL构建成功。结晶紫染色结果表明,Ad.TERT-TRAIL对肿瘤细胞的杀伤力比Ad.TERT高10～100倍,而对正常细胞的影响跟Ad.TERT一样小。100M OI(m ultiple ofinfection)Ad.TERT-TRAIL感染结肠癌细胞SW62072h后,细胞存活率为4%;而感染了Ad.TERT的SW620细胞存活率却达56%。两种病? BACKGROUND & OBJECTIVE: Some scholars have used the human telomerase reverse transcriptase (TERT) promoter to replace the native adenovirus E1A promoter to construct a tumor-specific proliferating virus, the oncolytic adenovirus Ad .TERT. Cell experiments and animal experiments show that, Ad.TERT has a good anti-cancer effect. The purpose of this study is to insert the apoptotic gene trail into Ad.TERT to generate Ad.TERT-TRAIL, to observe whether it has a stronger efficacy of killing tumor cells and to study its mechanism. METHODS: The Ad.TERT-TRAIL virus was constructed by homologous recombination of the adenovirus packaging plasmid pBH GE3 and the pZhTERT-trail carrying the tracer in H EK293 cells, and then identified by PCR. The expression of E1A and trail genes was detected by Western blot To further confirm. The crystal violet staining method and M TT method were used to detect the difference between the killing effect of Ad.TERT and Ad.TERT-TRAIL on tumor cells and normal cells. The expression of Caspase-3 was detected by Western blot to observe the apoptosis of tumor cells, and the apoptosis rate of tumor cells was determined by flow cytometry. Results: The band amplified by PCR was 860bp, which was the trail gene. Western blot results showed that E1A and trail in Ad.TERT-TRAIL were only expressed in tumor cells, indicating that the construction of Ad.TERT-TRAIL was successful. Crystal Violet staining showed that Ad.TERT-TRAIL on the tumor cell killing force than Ad.TERT 10 to 100 times higher, while on normal cells with Ad.TERT as small. 100M OI (m ultiple ofinfection) The survival rate of SW62072h cells infected with Ad.TERT-TRAIL was 4%, while the survival rate of SW620 cells infected with Ad.TERT was 56%. Two kinds of disease?