靶向性蛋白激酶B1和环氧合酶-2shRNA腺病毒载体的构建和在人胃癌细胞的表达  

Construction of a shRNA adenovirus vector targeting protein kinase B1 and cyclooxygenase-2 and expression in human gastric carcinoma cells

作　　者：张靖[1] 付彦超[1] 康春生[1] 张庆瑜[1] 

机构地区：[1]天津医科大学总医院消化科,300052

出　　处：《中华实验外科杂志》2009年第7期880-883,共4页Chinese Journal of Experimental Surgery

基　　金：天津市应用基础重点资助项目（07JCZDJC07700）

摘　　要：目的构建靶向性蛋白激酶B1（PKB1／Akt1）和环氧合酶-2（COX-2）的短发夹RNA（shRNA）腺病毒载体，观察其在人胃癌细胞株SGC-7901中的表达。方法利用同源重组技术构建重组腺病毒载体pGSadeno—Akt1＋COX-2（pGSadeno—A＋C），经酶切及测序鉴定后转染人胚肾细胞HEK293包装成为重组rAd5-A＋C腺病毒，并测定病毒的滴度。体外转染人胃癌细胞株SGC-7901后，Real—timePCR和蛋白印迹分别检测Akt1和COX-2mRNA和蛋白质的表达。结果成功构建rAd5．A＋C重组腺病毒，测定包装的病毒滴度为1．0×10^10pfu／ml。转染组Akt1和COX-2mRNA表达明显下调，其ACt值分别是（12．26±0．05）和（5．41±0．09），比rAd5-HK空载组（10．63±0．02）、（3．75±0．08）和对照组（10．57-1-0．02）、（3．73±0．08）增高（P〈0．01），而空载组和对照组比较ACt值无明显变化（P〉0．05）。同样转染组Akt1和COX-2蛋白表达量与空载组和对照组比较分别下调70．5％和63．7％（P〈0．01），空载组和对照组比较Akt1和COX-2蛋白表达差异无统计学意义（P〉0．05）。结论靶向性Akt1和COX-2的shRNA腺病毒载体可以特异性抑制Akt1和COX-2的表达，可能成为胃癌靶向性Akt1和COX-2基因治疗的新策略。Objective To construct a short hairpin RNA （shRNA） adenovirus vector targeting protein kinase B1 （PKB1/Akt1） and cyclooxygenase-2 （COX-2） and observe their expression in human gastric carcinoma cell line SGC-7901. Methods Aktl and COX-2 shRNA expression frames were subcloned to pGSadeno adenovirus vector by homologous recombination technology to construct pGSadeno- Akt1 ± COX-2 （pGSadeno-A ± C） vector. Furthermore after screening and amplification,recombinant adenovirus vector was digested with PacI and transfected into HEK293 cells. The replication adenovirus rAd5-A ± C was packed and amplified in the HEK293 cells, and its titer was detected. After human SGC-7901 cells in vitro were transfected by rAd5-A ± C ,Aktl and COX-2 mRNA and protein expression levels were detected by real-time PCR and Western blot respectively. Compared with rAdS-A ± C,SGC-7901 and general rAdS-HK were selected as the negative controls. Results The recombinant adenovirus rAdS-A ± C was constructed successfully and its titer reached 1.0 × 10^l0 pfu/ml. Aktl and COX-2 mRNA expression was downregulated significantly,and their ACt values （ 12.26 ±0.05 and 5.41 ±0.09 respectively） were higher than rAdS-HK group （ 10.63 -± 0.02 and 3.75 ± 0.08 respectively） and control group （ 10.57 ± 0.02 and 3.73± 0.08 respectively） （P 〈 0.01 ）. There was no significant difference between rAd5-HK and control groups （ P 〉 0.05 ）. Aktl and COX-2 protein expression was downregulated by 70.5% and 63.7% respectively （P 〈0.01 ） in rAd5-HK group as compared with control group （P 〉0.05）. Conclusion The shRNA adenovirus vector targeting Akt1 and COX-2 can specifically inhibit Akt1 and COX-2 expression, and this may be a new strategy in gastric carcinoma gene therapy targeting Akt1 and COX-2.

关 键 词：胃癌 蛋白激酶B1 环氧合酶2 短发夹RNA 腺病毒载体 

分 类 号：R735[医药卫生—肿瘤]