CAJ | 학술논문

目的构建能选择性杀伤表达癌胚抗原(CEA)的结直肠癌细胞的条件复制型腺病毒载体.方法 PCR扩增CEA基因5'端调控序列,构建以EGFPLuc作为报告基因的表达质粒pCEA-EGFPLuc.利用脂质体将pCEA-EGFPLuc导入CEA阳性和阴性细胞中,通过检测增强型绿色荧光蛋白(EGFP)表达和荧光素酶(luciferase)活性,评价CEA启动子活性.构建由CEA启动子调控的E1A,并携带HSVtk基因的条件复制型腺病毒Ad.CEA-E1A/CMV-TK,分别感染CEA阳性肿瘤细胞(Lovo和SW620)和阴性肿瘤细胞(HeLa),通过E1A表达和细胞存活率检测,评价该病毒对CEA阳性肿瘤细胞的选择性杀伤效果及联合应用环氧鸟苷(GCV)后的协同效应.结果 CEA启动子具有较好的选择性和较高的活性.Ad.CEA-E1A/CMV-TK只在CEA阳性的肿瘤细胞中表达E1A.感染Ad.CEA-ElA/CMV-TK后,Lovo和SW620细胞的存活率分别为(36.72±2.49)%和(39.82±4.76)%,均显著低于HeLa细胞的(87.44±2.76)%(P<0.01);联合GCV对Lovo和SW620的杀伤效果增加,细胞存活率分别为(17.26±3.65)%和(23.93±5.40)%,显著低于未加GCV的(36.72±2.49)%和(39.82±4.76)%(P<0.01).结论由CEA调控复制的Ad.CEA-E1A/CMV-TK对CEA阳性的结直肠癌细胞具有选择性杀伤作用,联合GCV后杀伤效果明显增强.
목적 구건능선택성살상표체암배항원(CEA)적결직장암세포적조건복제형선병독재체.방법 PCR확증CEA기인5'단조공서렬,구건이EGFPLuc작위보고기인적표체질립pCEA-EGFPLuc.이용지질체장pCEA-EGFPLuc도입CEA양성화음성세포중,통과검측증강형록색형광단백(EGFP)표체화형광소매(luciferase)활성,평개CEA계동자활성.구건유CEA계동자조공적E1A,병휴대HSVtk기인적조건복제형선병독Ad.CEA-E1A/CMV-TK,분별감염CEA양성종류세포(Lovo화SW620)화음성종류세포(HeLa),통과E1A표체화세포존활솔검측,평개해병독대CEA양성종류세포적선택성살상효과급연합응용배양조감(GCV)후적협동효응.결과 CEA계동자구유교호적선택성화교고적활성.Ad.CEA-E1A/CMV-TK지재CEA양성적종류세포중표체E1A.감염Ad.CEA-ElA/CMV-TK후,Lovo화SW620세포적존활솔분별위(36.72±2.49)%화(39.82±4.76)%,균현저저우HeLa세포적(87.44±2.76)%(P<0.01);연합GCV대Lovo화SW620적살상효과증가,세포존활솔분별위(17.26±3.65)%화(23.93±5.40)%,현저저우미가GCV적(36.72±2.49)%화(39.82±4.76)%(P<0.01).결론 유CEA조공복제적Ad.CEA-E1A/CMV-TK대CEA양성적결직장암세포구유선택성살상작용,연합GCV후살상효과명현증강.
Objective To construct a new conditionally replicative adenovirus (CRAds) targeting carcinoembryonic antigen (CEA) positive colorectal cancer cells. Methods The DNA fragment of the CEA gene promoter was amplified through PCR and cloned into the vector carrying fusion reporter gene EGFP-Luc to construct expression plasmid pCEA-EGFPLuc. The constructed plasmid pCEA-EGFPLuc was transfected into CEA positive and negative cells by liposome. The activity of CEA gene promoter was evaluated by detecting the expression of EGFP and luciferase activity. The conditionally replicative adenovirus Ad.CEA-E1A/CMV-TK carrying suicide gene HSVtk was constructed, in which the E1A gene was controlled under CEA promoter. CEA positive(Lovo and SW620)and negative tumor cells(HeLa) were infected with Ad.CEA-E1A/ CMV-TK. The selective cytotoxicity of Ad.CEA-E1A/CMV-TK and the synergistic effect of the virus with GCV in CEA positive tumor cells were evaluated by the expression of E1A, cytopathic effect and cell survival rate. Results CEA promoter possesses a good specificity as well as high activity. The expression of E1A only presented in CEA positive tumor cells. After infection with Ad. CEA-E1A/CMV-TK, the cell survival rates of Lovo and SW620 were (36.72±2.49)% and (39.82±4.76)%, respectively, significantly lower than that of Hela[(87.44±2.76)%1 (P<0.01). When combined with GCV, Ad.CEA-E1A/CMV-TK had better oncolytic effect on Lovo and SW620 cells, with cell survival rates of (17.26±3.65) % and (23.93±5.40) %, respectively, significantly lower than those without GCV[(36.72±2.49) % and (39.82±4.76) %, respectively] (P<0.01). Conclusion Ad. CEA-E1A/CMV-TK under the control of CEA promoter has selective cytotoxic effect on CEA positive colorectal cancer cells, and the effect can be enhanced when combined with GCV.