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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 investigate the specificity of human pancreatic amylase gene 2 (Amy-2) promoter in human pancreatic tissues and to verify its activity in pancreatic cells. Methods Amy-2 plasmid was constructed by using luciferase reporter vector and transfected into human pancreatic cancer cell lines SW1990, PANC-1, normal human pancreatic ductal epithelial cells (HPDE), breast cancer (MCF-7 ), Lung cancer (A549), to verify its activity and specificity in pancreatic cells. The pAmy2-Cre vector was constructed by using molecular biology techniques combined with PBS185 vector. The pAmy2-Cre was transfected into human pancreatic cancer cell lines Panc-1 and BxPC-3 by gene transfection method. The expression of pAmy2- 2 tissue specificity. Results The pAmy-2-luc and pAmy-2-Cre vectors were successfully constructed and proved to be transcriptional activators of Amy-2 in corresponding cells. The promoter of Amy-2 can drive the expression of Cre protein. Conclusion Amy-2 has better pancreatic tissue specificity and transcription.