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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。
Objective To obtain human TIMP-2 protein by prokaryotic expression and to explore the role of TIMP-2 in the migration and invasion of hepatocellular carcinoma cells. METHODS: Human TIM P-2 gene cDNA was amplified by RT-PCR using total RNA from SMCC 7721 hepatoma cells as a template. The prokaryotic expression vector pGEX-TIMP-2 was constructed by gene recombination and transfected into E. coli Origami (DE3) to induce the expression of human TIMP-2 recombinant protein. The expressed products were purified by affinity chromatography and identified by Western blotting. The effect of TIMP-2 protein on the migration and invasion of hepatocellular carcinoma cells was analyzed by RNAi and addition of foreign proteins. The expression of siRNA-TIMP-2 The mRNA and protein expression of endogenous TIMP-2 in hepatocellular carcinoma cells after transfection. Results The exogenously added TIMP-2 protein inhibited the migration and invasion of hepatocellular carcinoma cells. However, RNAi inhibited the migration and invasion of hepatocellular carcinoma cells by endogenous TIMP-2 protein. The conditioned medium of hepatic stellate cells could promote the growth of liver cancer Cell migration and invasion, and this effect can be inhibited by the TIMP-2 protein. Conclusion The TIMP-2 protein level in HCC microenvironment is closely related to the migration and invasion of HCC cells.