Amy-2启动子的克隆和活性鉴定  

Construction of promoter Amy-2 and identification of its tissue-specific activity in pancreatic cells

作　　者：周家华[1] 唐波[2] 刘训良[3] 丁帆[2] 何道伟[4] 何勇[5] 余泽前[1] 杨德同[1] 

机构地区：[1]东南大学附属中大医院,南京210009 [2]南京大学国家生物医药重点实验室 [3]南京医科大学第一附属医院 [4]东南大学临床医学院 [5]东南大学遗传中心

出　　处：《江苏医药》2009年第12期1479-1482,共4页Jiangsu Medical Journal

基　　金：南京市科研基金(200801082);东南大学科技基金资助(XJ0690219)

摘　　要：目的探讨人胰腺淀粉酶基因2(Amy-2)启动子在人胰腺组织特异性并验证其在胰腺细胞内的活性。方法运用荧光素酶报告载体构建胰腺组织特异性启动子Amy-2质粒,将其转染人胰腺癌细胞系SW1990、PANC-1、正常人胰腺导管上皮细胞(HPDE)、乳腺癌(MCF-7)、肺癌(A549),验证其在胰腺细胞内的活性和特异性。利用分子生物学技术,结合PBS185载体构建载体pAmy2-Cre;在运用基因转染方法将pAmy2-Cre的转染人胰腺癌细胞株Panc-1、BxPC-3,通过PCR、Western blot方法观察pAmy-2的组织特异性。结果成功构建pAmy-2-luc和pAmy-2-Cre载体,证明了Amy-2的启动子在相应细胞内的转录活性;Amy-2的启动子能很好的驱动Cre蛋白表达。结论Amy-2有较好的胰腺组织特异性和转录。Objective To examine the efficacy of gene under the control of pancreatic-tissuespecific promoter of human amylase-2 gene （Amy-2）. Methods Transcriptional activities of Amy-2 promoter sequences were analyzed using a luciferase reporter gene on human pancreatic cancer cell lines SW1990 and PANC-1, human pancreatic duct epithelial cell （HPDE）, human breast cancer cell line MCF, human lung cancer cell line A549 cell lines. The recombinant pAmy-2-Cre vector was constructed with PBS185 vector. The efficacy of pAmy-2-Cre on the transiently tranefected Pane-1 and BxPC-3 human pancreatic cancer cell lines. Expression of Cre gene of transiently trancfected cells was measured by Western blot assay in vitro. Results The Amy-2 promoter had high transcriptional activity in transfected tumor cell-lines and pancreas-derived cells respectively. Expression of Cre gene with pAmy-2-Cre vector was proved to be tissue-specific promoter Amy-2 regulated. Conclusion The promoter Amy-2 has high transcriptional activities and tissue-specific activity in pancreatic cancer cells.

关 键 词：人胰腺淀粉酶基因2 胰腺癌 

分 类 号：Q343[生物学—遗传学]