胰腺癌特异性受体基因载体的构建及环氧合酶2L启动子的作用  

Construction of gene vector containing somatostatin receptor type Ⅱ gene and function of cyclooxygemine-2 promoter

作　　者：王东[1] 巩鹏[1] 王忠裕[1] 

机构地区：[1]大连医科大学附属第一医院普外二科,辽宁116011

出　　处：《中华实验外科杂志》2006年第11期1322-1324,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(30471703)

摘　　要：目的构建环氧合酶-2L启动子(Cox-2L)及2型生长抑素受体(SSTR2)基因重组腺病毒载体,以研究SSTR2重新表达及腺病毒感染对胰腺癌细胞的影响。方法用逆转录-聚合酶链反应(RT-PCR)法从人正常胰腺组织中调取SSTR2基因,以HL60细胞基因组DNA为模板,以PCR法调取Cox-2L基因,以SSTR2及Cox-2L基因片段为模板,以PCR法扩增Cox-2L-SSTR2融合基因并连入TaKaRa公司pAxcwit腺病毒载体中,以电穿孔法转化E．Coli后挑选阳性克隆进行PCR及酶切鉴定。以pAxcwit-Cox-2L-SSTR2转染HEK293细胞获得重组腺病毒颗粒,进行PCR、酶切鉴定。结果调取并连接后的基因片段经测序证实为Cox-2L、SSTR2及Cox-2L-SSTR2融合基因,Cox-2L-SSTR2基因片段及PolyA克隆入T载体,经酶切并测序证实PMDl8-T-Cox-2L- SSTR2-PolyA构建成功。pAxcwit-Cox-2L-SSTR2-PolyA经酶切及PCR鉴定,Cox-2L-SSTR2-PolyA融合基因正确连入腺病毒载体。结论成功构建了Cox-2L-SSTR2基因重组腺病毒载体,为进一步研究SSTR2在胰腺癌细胞中的表达及作用奠定了基础。Objective To construct recombinant adenoviral vector containing Cox-2L promoter and SSTR2 gene so as to observe the effects of SSTR2 re-expression and adenovirus infection on the growth of pancreatic cancer cells. Methods Human SSTR2 cDNA was amplified by RT-PCR from normal human pancreas tissue; Cox-2L DNA was amplified by PCR from human HL 60 cells. Cox-2L-SSTR2 fusion gene was amplified by PCR and the pAxcwit Adenoviral Vector System was used to generate replication defective adenoviral vector carrying Cox-2L-SSTR2 fusion gene. The recombinant adenoviral vectors were confirmed by PCR and restriction enzyme digestion analysis. The pAxcwit Cox-2L-SSTR2 was transfected into HEK293 cell through lipofectamine and identified by PCR and restriction enzyme digestion analysis. Results The three fragments were confirmed to be Cox-2L gene,SSTR2 gene and Cox-2L- SSTR2 fusion gene by sequence analysis. The gene fragments of Cox-2L-SSTR2 and PolyA were cloned into PMD18-T vector to construct PMDI8-T-Cox-2L-SSTR2-PotyA and then confirmed by restriction enzyme digestion and sequence analysis. The constructed recombinant adenoviral vector was detected by PCR and restriction enzyme digestion to confirm that Cox-2L-SSTR2-PolyA fusion gene was correctly cloned into adenoviral vector. Conclusion The recombinant adenoviral vector containing Cox-2L and SSTR2 genes was constructed successfully. This results lays the foundation for further study on function of SSTR2 in pancreatic cancer cell.

关 键 词：启动子 生长抑素受体 胰腺癌 

分 类 号：R735.9[医药卫生—肿瘤]