TFPI-2基因克隆及其在胰腺癌细胞中的表达  

Molecular Cloning of Human Tissue Factor Pathway Inhibitor 2 and Its Expression in Panc-1 Cells

作　　者：汤志刚[1] 孙振阳[1] 胡何节[1] 许戈良[1] 陈炯[1] 李建生[1] 

机构地区：[1]安徽医科大学附属安徽省立医院普通外科,合肥230001

出　　处：《肿瘤防治研究》2008年第1期5-7,共3页Cancer Research on Prevention and Treatment

基　　金：安徽省自然科学基金资助项目(050430702)

摘　　要：目的克隆人TFPI-2基因全长cDNA,构建其真核表达载体,并将其转染到人胰腺癌细胞Panc-1中,检测其表达。方法用RT-PCR法从人胎盘组织中扩增人TFPI-2基因,并将其与真核表达载体pEGFP-C1连接,构建真核表达载体pEGFP-C1-TFPI-2。将构建的重组载体转染到人胰腺癌细胞系Panc-1细胞,Western blot检测TFPI-2在Panc-1细胞中的表达。结果RT-PCR成功的扩增出一条约708bp的片断,扩增片断与载体连接后,经限制性内切酶酶切电泳分析和DNA序列测定证实该基因已经成功构建到载体上,转染Panc-1细胞后,荧光显微镜观察到稳定转染细胞发出较强绿色荧光,Western blot技术证明TFPI-2基因能在Panc-1细胞中稳定表达。结论成功构建了人TFPI-2基因的真核表达载体pEGFP-C1-TFPI-2,获得了稳定表达TFPI-2的Panc-1细胞,证实TFPI-2基因能够在人胰腺癌细胞系Panc-1中高效、稳定的表达,为进一步研究其胰腺癌迁移、浸润及转移中的作用打下基础。Objective To clone the total length cDNA of TFPI-2 gene and construct its eukaryotic expression vector. Then the eukaryotic expression vector was transfected into pancreatic carcinoma cell line Panc-1, the expression of TFPI-2 was detected. Methods The TFPI-2 gene was amplified by RT-PCR from human placenta tissue and was inserted into eukaryotic expression vector pEGFP-C1, then the eukaryotic expression vector pEGFP-C1-TFPI-2 was constructed. The recombinant plasmid was then transfected into pancreatic carcinoma cell line Panc-1 cells. The expression of TFPI-2 in transfected cells was detected by Western blot. Results By the use of RT-PCR, the fragment of 708 bp of TFPI-2 gene was successfully amplified. After being inserted into the vector, enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into recombinant vector successfully. After being transfected into Pane-1 cell, green fluorescence was emitted from transfected cells under fluorescent microscope. Western blot analysis revealed that the TFPI-2 gene could be expressed stably in the transfected cells. Conclusion The eukaryotic expression vector pEGFP-C1-TFPI-2 was successfully constructed. The Panc-1 cell that could express TFPI-2 stably was obtained. It was also confirmed that the TFPI-2 gene could be expressed in Panc-1 cell with high-performance and stability. These established a foundation for further study of TFPI-2 in the invasion and migration of pancreatic carcinoma cells.

关 键 词：胰腺肿瘤 真核表达载体 组织因子途径抑制物2(TFPI-2) 

分 类 号：R735.9[医药卫生—肿瘤] Q785[医药卫生—临床医学]