携带人肝细胞生长因子第一结构域基因溶瘤腺病毒的构建及其对胃癌细胞的杀伤作用  

Construction of oncolytic adenovirns carrying kringle 1 domain of hepatocyte growth factor gene and its lethal effect to gastric cancer cells

作　　者：巢晨 马贵亮[1] 任红[1] 毛伟征[1] 

机构地区：[1]南京医科大学青岛临床学院青岛市立医院普外中心,青岛266071

出　　处：《中华实验外科杂志》2017年第6期973-975,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金（81272519）

摘　　要：目的构建携带具有抗肿瘤细胞和抗血管生成作用的人肝细胞生长因子第一结构域（HGFK1）基因的肿瘤特异性启动子调控的溶瘤腺病毒，研究其对胃癌细胞选择性杀伤作用。方法以人端粒酶反转录酶启动子（hTERT）和缺氧调控元件序列（HRE）分别调控溶瘤腺病毒复制必要的E1a和E1b基因，基因组插入HGFK1基因，构建HGFK1溶瘤腺病毒（TH—Ad—HGFK1-EGFP）和无HGFK1溶瘤腺病毒（TH—Ad—EGFP），空斑计数法滴定病毒浓度；病毒分为3组：实验A组（TH-Ad—HGFK1-EGFP）、实验B组（TH-Ad—EGFP）和对照C组（Ad-EGFP），分别感染胃癌细胞SGC7901和正常人成纤维细胞（HF）；半数细胞培养物感染量法（TCID50）检测病毒扩增倍数；噻唑蓝（MTT）法检测病毒杀伤抑制作用；双重荧光染色检测病毒促凋亡作用。结果溶瘤腺病毒TH—Ad—HGFK1-EGFP、TH-Ad—EGFP经测序及聚合酶链反应（PCR）鉴定确认构建成功，滴度都为2×10^10pfu／ml；病毒扩增结果显示：A、B组病毒在SGC7901细胞中的扩增倍数分别为1．47×10^4、1．60×10^4倍，远大于对照C组病毒，在HF细胞中为110倍和124倍，远小于对照C组病毒；MTF结果显示：当感染复数（MOI）=100时，A组病毒对SGC7901细胞72、96h后的抑制率为（71．34±8．27）％、（74．56±1．78）％，B组为（30．58±3．83）％、（31．84±6．62）％，C组为（23．40±2．29）％、（21．18-4-2．32）％。A组与B组两时间点分别比较差异有统计学意义（P=0．002、P=0．003），A组与C组分别比较差异有统计学意义（P=0．003、P=0．000）。A、B组病毒对HF细胞在96h后的抑制率为（18．94±0．88）％、（22．84±3．34）％，C组为（53．25±3．50）％，A、B两组与C组分别比较差异有统计学意义（P=0．003、P=0．001）；凋亡结果显示：A组SGC7901细胞的凋亡率为（18．66±4．04）％，远高于其余各组。结论溶瘤腺病�Objective To construct an double -regulated oncolytic adenovirus carrying kringle 1 domain of hepatocyte growth factor （ HGFK1 ） gene which exerts anti - tumor and anti - angiogenesis effect and observe its lethal effect to gastric cancer cells. Methods Adenoviral Ela and Elb genes were con- trolled by Human telomerase reverse transcriptase promoter （hTERT） and hypoxia regulatory element （HRE）. TH - Ad - HGFK1 - EGFP or TH - Ad - EGFP was constructed by inserting HGFK1 gene into the viral genome or not. The adenovirus was divided into three groups： the experimental group A （TH- Ad- HGFK1 -EGFP）, the experimental group B （TH -Ad -EGFP） and the control group C （Ad - EGFP）. Gastric cancer celts SGC7901 and the normal human fibroblasts cell HF were infected by adenovirus, then the proliferation of adenovirus was counted by the half - cell culture infection dose （TCIDS0） method. Methyl thiazolyl tetrazolium （MTr） method was used to detect the inhibitory effect. 4, 6 - Diamidino - 2 - Phenylindole, Dihydrochloride （DAPI） method and Terminal - deoxynucleoitidyl Transferase Mediated Nick End Labeling （TUNEL） method was used to observe the cell apoptosis. Results TH - Ad - HGFK1 - EGFP and TH - Ad - EGFP was confirmed constructed successfully by DNA sequen- cing and PCR analyses. All the titer of the adenovirus was 2 ×10^10 pfu/ml. The TCID50 results showed the proliferation of group A and group B in SGC7901 cells was 1.47 ×10^4 and 1.60 ×10^4 fold, which were sig- nificantly higher than that in group C. However in HF cells, the fold change of replication of group A and group B was only 110 and 124, which was significantly lower than that in group C. Group A showed potent inhibitory effect on the growth of SC7901 cells, but Group B and group C showed no obviously inhibitory effect. The inhibition ratio of group A （MOI = 100, 72 h and 96 h） on SGC7901 cells was （71.34 ± 8.27 ）% , （74. 56 ± 1.78）% , which had significant difference compared with those in group B （

关 键 词：胃癌 人肝细胞生长因子第一结构域 溶瘤腺病毒 SGC7901 

分 类 号：R735.2[医药卫生—肿瘤]