中文摘要：

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SM M C-7721肝癌细胞总RNA为模板,用RT-PCR技术扩增出人TIM P-2基因c DNA,通过基因重组技术构建原核表达载体p GEX-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。

英文摘要：

Objective To collect protein of human TIM P-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 SM M C-7721,and TIM P-2 c DNA was obtained with RT-PCR. The prokaryotic expression vector p GEX-TIM P-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 TIM P-2 in migration and invasion of HCC cells were detected with RNAi technology and adding extraneous TIM P-2 protein. The mRNA and protein expressions of endogenous TIM P-2 in HCC cells were detected with real-time fluorescence quantitative PCR and Western blotting after transfection with si TIM P-2. Results Adding of exogenous TIM P-2 suppressed the migration and invasion of HCC; knock-down of TIM P-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 TIM P-2 protein. Conclusion The level of TIM P-2 protein in the microenvironment is closely related to the migration and invasion of HCC.