人TIMP-2蛋白在肝癌细胞迁移与侵袭中的作用

doi:10.6040/j.issn.1671-7554.0.2016.132

摘要/Abstract

摘要： 目的通过原核表达获取人TIMP-2蛋白,探讨TIMP-2在肝癌细胞迁移及侵袭中的作用。方法以SMMC-7721肝癌细胞总RNA为模板,用RT-PCR技术扩增出人TIMP-2基因cDNA,通过基因重组技术构建原核表达载体pGEX-TIMP-2并在大肠杆菌Origami(DE3)中诱导表达人TIMP-2重组蛋白。采用亲和层析法纯化表达产物,并用Western blotting鉴定;用RNAi技术和添加外源蛋白方法分析TIMP-2蛋白对肝癌细胞迁移和侵袭的作用;荧光定量PCR 、Western blotting检测siRNA-TIMP-2转染细胞后肝癌细胞内源性TIMP-2的mRNA及蛋白表达水平。结果外源添加的TIMP-2 蛋白抑制肝癌细胞迁移及侵袭的能力,而RNAi技术能抑制内源的TIMP-2蛋白增强肝癌细胞迁移及侵袭的能力;肝星状细胞的条件培养液能够促进肝癌细胞迁移和侵袭,而这种作用能够被TIMP-2蛋白抑制。结论肝癌微环境中的TIMP-2蛋白水平与肝癌细胞的迁移和浸润密切相关。

关键词: 肝细胞癌, 侵袭, 转移, 基质金属蛋白酶抑制剂2

Abstract: Objective To collect protein of human TIMP-2 through prokaryotic expression in vitro, and to explore its role in the migration and invasion of hepatocellular carcinoma(HCC)cell. Methods Total RNA was extracted from human hepatoma cell line SMMC-7721, and TIMP-2 cDNA was obtained with RT-PCR. The prokaryotic expression vector pGEX-TIMP-2 was constructed, and then transformed into Origami(DE3). The expressional product was purified with affinity chromatography and identified with Western blotting. The role of TIMP-2 in migration and invasion of HCC cells were detected with RNAi technology and adding extraneous TIMP-2 protein. The mRNA and protein expressions of endogenous TIMP-2 in HCC cells were detected with real-time fluorescence quantitative PCR and Western blotting after transfection with siTIMP-2. Results Adding of exogenous TIMP-2 suppressed the migration and invasion of HCC; knock-down of TIMP-2 enhanced the migration and invasion of HCC; conditioned medium of hepatic stellate cells promoted the migration and invasion of HCC, which could be inhibited by TIMP-2 protein. Conclusion The level of TIMP-2 protein in the microenvironment is closely related to the migration and invasion of HCC.

Key words: Tissue inhibitor of matrix metalloproteinases-2, Invasion, Hepatocellular carcinoma, Migration

中图分类号:

R735.7

王伟,曹煜姗,孙达权,黄小琼,徐国强. 人TIMP-2蛋白在肝癌细胞迁移与侵袭中的作用[J]. 山东大学学报（医学版）, 2016, 54(7): 11-17.

WANG Wei, CAO Yushan, SUN Daquan, HUANG Xiaoqiong, XU Guoqiang. Role of human TIMP-2 protein in the migration and invasion of hepatocellular carcinoma cell[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2016, 54(7): 11-17.

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