pcDNA3.1-KISS-1真核表达载体的构建及其对EC1细胞转移的抑制作用  

Construction of Eukaryotic Expression Plasmid pcDNA3.1-KISS-1 and Its Metastasis Inhibiting Effect to EC1 Cells

作　　者：李娜[1,2] 李珊珊[1] 张红艳[1] 轩小燕[1] 郑献召[1] 王丰[1] 闫爱华[1] 

机构地区：[1]郑州大学第一附属医院病理科,450052 [2]新乡医学院病理教研室,453003

出　　处：《肿瘤防治研究》2009年第4期265-269,共5页Cancer Research on Prevention and Treatment

基　　金：河南省科技攻关基金资助(072102310042)

摘　　要：目的克隆人食管组织KISS-1基因及构建真核表达载体pcDNA3.1-KISS-1,并观察其在食管癌EC1细胞中的表达及对EC1细胞侵袭及运动能力的影响。方法从人正常食管组织中提取总RNA,设计特异性引物,通过逆转录-聚合酶联链反应(RT-PCR)扩增KISS-1基因cDNA序列,并将其克隆到真核表达载体pcDNA3.1中,构建真核表达质粒pcDNA3.1-KISS-1,酶切鉴定及测序分析后用Lipofectamine脂质体将其转染到食管癌细胞,Westernblot法检测其蛋白在EC1细胞中的表达情况;应用细胞运动实验及重建基底膜侵袭实验分析KISS-1表达对EC1细胞运动和侵袭能力的影响。结果成功克隆了人KISS-1基因全长编码序列,构建了重组真核表达载体pcDNA3.1-KISS-1,脂质体转染后KISS-1基因在EC1细胞中成功表达;转基因组细胞的运动能力(174.6±14.24)和体外穿越重建基底膜的能力(90.44±12.83)明显低于转空质粒组(222.6±30.08,110.22±15.87)和对照组EC1细胞(234.40±14.72,124.78±18.14)(P均<0.05)。结论重组pcDNA3.1-KISS-1真核表达质粒构建成功,并且KISS-1对食管癌细胞的侵袭和运动能力具有抑制作用。Objective To clone human esophageal tissue KISS-1 gene and construct its eukaryotic expression vector pcDNA3. 1-KISS-1. To detect its expression in EC1 cells and observe the influence of transfection KISS-1 gene on the invasion and migration ability of EC1 cells. Methods Total RNA was extracted from human esophageal tissue. KISS-1 cDNA was isolated by using RT-PCR,and cloned into the eukaryotic expression vector pcDNA3. 1. The eukaryotic expression plasmid pcDNA3. 1-KISS-1 was verified by sequencing and enzyme digestion analysis. Using liposomemediated transfection technique, the eukaryotic expression vector pcDNA3. 1-KISS-1 was transfected into EC1 cells. The expression of protein in EC1 cells was detected by Western blot. The effect of KISS-1 expression on the invasion and migration of EC1 cells was investigated by Boyden-chamber system. Results pcDNA3. 1-KISS-1 recombinant plasmid was successfully constructed and it had a higher level expressed in ECI cells. Matrigel invasion and migration assay showed that the invasion and migration of cells transfected with KISS-1 were less than those in cells transfected with pcDNA3.1 and control cells （ P 〈 0. 05 ）. Conclusion The expression of KISS-1 could inhibit EC1 ceils invasion and migration in vitro.

关 键 词：KISS-1基因 基因克隆 真核表达载体 食管癌细胞株 

分 类 号：R735.1[医药卫生—肿瘤]