缺氧反应元件嵌合特异性启动子调控的溶瘤腺病毒对肾癌细胞的杀伤作用  

Antitumor effect of hypoxia response element chimeric tumor specific promoter regulated oncolytic adenovirus on renal cell carcinoma

作　　者：秦静 张晨 田伟平 陈静 方琳 Qin Jing;Zhang Chen;Tian Weiping;Chen Jing;Fang Lin(Cancer Institute,Xuzhou Medical University,Xuzhou 221002,China;Center of Clinical Oncology,Affiliated Hospital of Xuzhou Medical University,Xuzhou 221002,China)

机构地区：[1]徐州医科大学肿瘤研究所,江苏221102 [2]徐州医科大学附属医院肿瘤科,江苏221102

出　　处：《中华实验外科杂志》2020年第1期90-92,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金面上项目(81872488);江苏省自然科学基金面上项目(BK20161156)。

摘　　要：目的观察低氧条件下缺氧反应元件(HRE)与肿瘤特异性细胞增殖相关核抗原Ki-67(MKI67)启动子构成的嵌合型启动子调控的溶瘤腺病毒在肾癌细胞中的增殖能力和杀伤作用。方法将HRE序列作为增强子嵌合至肿瘤特异性强的Ki-67启动子上游,同时携带报告基因增强型绿色荧光蛋白(EGFP),构建溶瘤腺病毒Ad-HRE-Ki-67-EGFP,组织半数感染剂量法(TCID50)测定滴度;两种病毒分别在常氧(21%O2)和低氧(1%O2)条件下感染正常肾上皮细胞人肾小管上皮细胞(HK-2)和肾癌细胞人肾细胞腺癌细胞(OSRC-2)、人肾腺癌细胞(ACHN),24 h后荧光显微镜及流式细胞仪检测病毒的靶向性;蛋白质印迹法(Western blot)检测低氧条件下细胞中低氧相关蛋白缺氧诱导因子(HIF)-1α、血管内皮生长因子(VEGF)的表达;细胞计数试剂盒(CCK-8)检测病毒对细胞的杀伤能力。应用SPSS 19.0统计软件分析,组间均数比较采用t检验。结果成功构建重组溶瘤腺病毒Ad-HRE-Ki-67-EGFP;Ad-HRE-Ki-67-EGFP感染肾癌细胞ACHN和OSRC-2,常氧条件下Ad-HRE-Ki-67-EGFP的感染效率分别为(47.5±2.6)%和(35.2±3.4)%,低氧条件下分别为(86.4±3.4)%和(73.5?±2.3)%,即低氧条件下感染效率更高(ACHN:t=15.760;OSRC-2:t=16.190;P<0.01);正常肾小管上皮细胞HK-2在常氧条件下Ad-Ki-67-EGFP、Ad-HRE-Ki-67-EGFP两种病毒的感染效率为(22.4±3.1)%和(22.9±2.2)%,与肾癌细胞ACHN、OSRC-2比较(ACHN:(68.4±3.3)%和(72.1±2.9)%;OSRC-2:(56.1±3.1)%和(61.4±2.3)%),差异有统计学意义(Ad-Ki-67-EGFP:t=17.650、13.390;Ad-HRE-Ki-67-EGFP:t=23.680、21.430,P<0.01),说明2种腺病毒对肾癌细胞具有靶向性;Western blot结果显示低氧条件下缺氧诱导因子HIF-1α、血管内皮生长因子VEGF、p53蛋白的表达增多且表现为时间依赖性,说明低氧可以调节HIF-1α、VEGF、p53蛋白的转录及翻译水平;在低氧条件下,感染Ad-HRE-Ki-67-EGFP的两种肿瘤细胞的早期区域1A(E1A)蛋白表达水平明显高于Ad-Ki-67Objective To investigate the effect of chimeric promoter composed of hypoxia response element(HRE)and tumor specific cell proliferation-associated nuclear antigen(Ki-67)promoter on the replication ability of oncolytic adenovirus and its cytotoxic activity under hypoxic conditions.Methods The HRE sequence,as an enhancer,was inserted upstream of the Ki-67 promoter and,at the same time carried the reporter gene enhanced green fluorescent protein(EGFP)to construct oncolytic adenovirus Ad-HRE-Ki-67-EGFP,and the titer of adenoviruses was measured by the tissue culture infective dose(TCID50).Normal renal epithelial cells HK-2 and renal cancer cells OSRC-2 and ACHN were infected with two kinds of viruses under normoxic(21%O2)or hypoxic(1%O2)conditions respectively for 24 h,and the virus tumor-targeting was detected by fluorescence microscopy and flow cytometry.Western blotting was performed to detect the expression of hypoxia-related protein hypoxia-inducible factor-1 alpha(HIF-1α),vascular endothelial growth factor(VEGF)and the expression of E1A protein.Cell counting kit-8(CCK-8)assay was used to detect the cytotoxity of virus on cells.Results The recombinant oncolytic virus Ad-HRE-Ki67-EGFP was successfully constructed.Under normoxic conditions,the efficiency of ACHN and OSRC-2 infection by chimeric HRE oncolytic adenovirus in renal cancer cells was(47.5±2.6)%and(35.2±3.4)%,while that in hypoxic conditions was(86.4±3.4)%and(73.5±2.3)%,indicating that the infection efficiency was higher under hypoxic conditions(ACHN:t=15.760;OSRC-2:t=16.190;P<0.01);Normal renal tubular epithelial cells HK-2 were infected with Ad-Ki-67-EGFP or Ad-HRE-Ki-67-EGFP under normoxic conditions.The infection efficiency was(22.4±3.1)%and(22.9±2.2)%respectively,compared with that of ACHN and OSRC-2 cells[ACHN:(68.4±3.3)%and(72.1±2.9)%;OSRC-2:(56.1±3.1)%and(61.4±2.3)%],the difference was statistically significant(Ad-Ki-67-EGFP:t=17.650,13.390;Ad-HRE-Ki-67-EGFP:t=23.680,21.430,P<0.01),indicating that the two adenoviruses have a targeting

关 键 词：肾细胞癌 溶瘤腺病毒 缺氧反应元件 低氧 

分 类 号：R737[医药卫生—肿瘤]