【摘要】 目的:研究在端粒酶启动子驱动下表达绿色荧光蛋白(GFP)及5型腺病毒早期基因(E1A)基因的腺病毒Ad/hTERT-GFP-E1对大肠癌细胞的杀伤作用及其可能的机制。方法:将不同滴度Ad/hTERT-GFP-E1感染大肠癌及正常细胞,以巨细胞病毒(CMV)启动子驱动下表达GFP基因的腺病毒Ad/CMV-GFP作为载体对照,通过半数组织培养感染量(TCID50)法测定病毒滴度及体外复制能力,然后用MTT法及细胞克隆形成试验评价该病毒在体外对肿瘤细胞及正常细胞的杀伤作用,并对病毒感染后的细胞进行原位凋亡检测。结果:Ad/hTERT-GFP-E1能够在DLD1细胞内持续复制,GFP的表达能够对病毒的感染和复制起到监测作用;MTT结果显示该病毒对于结直肠肿瘤细胞有显著杀伤作用,而对于正常细胞没有明显的杀伤作用;对病毒感染后的DLD1进行细胞凋亡检测发现Ad/hTERT-GFP-E1所引起的凋亡率显著高于对照组(P<0.01)。结论:溶瘤腺病毒Ad/hTERT-GFP-E1能够选择性地在肿瘤细胞内复制并杀伤肿瘤细胞,其机制与细胞凋亡的途径有关。更多还原

【Abstract】 AIM: To investigate the killing effects and its mechanisms of the adenovirus in which the transgene green fluorescent protein(GFP) and adenovirus 5 early region 1A(E1A) were both under the control of human telomerase reverse transcriptase promoter on colon cells.METHODS: Colon cells and normal cells were treated with different titers of Ad/hTERT-GFP-E1,Ad/CMV-GFP in which the transgenes GFP was driven by CMV promoter as the vector control.We used TCID50 to detect replication titer in vitro.MTT assay and clonogenic assay were performed to determine the killing effect on the virus in vitro.In situ cell death detection kit was used to detect apoptosis level after treatment.RESULTS: The process of Ad/hTERT-GFP-E1 replication in DLD1 cells was monitored by detecting GFP expression.MTT assay demonstrated that Ad/hTERT-GFP-E1 had killing effect on colon cells,but no notable toxicity was evident in NHFB cells.Apoptosis assay after treatment showed that the percentage of apoptotic cells in Ad/hTERT-GFP-E1 group was obviously higher than that in control group(P<0.01).CONCLUSION: The oncolytic adenovirus Ad/hTERT-GFP-E1 has the ability of tumor specific replication and killing effect in cancer cells.The mechanism of this effect involves basic apoptotic pathway.更多还原