正、反义基质金属蛋白酶组织抑制因子-1基因对小鼠成纤维细胞表达细胞外基质的影响

Effects of Tissue Inhibitor of Metalloproteinase-1 on Cell Proliferation,matrix Metalloproteinases(MMPs) and Extracellular matrix(ECM) Degradation in Fibroblast

目的 观察正、反义基质金属蛋白酶组织抑制因子 - 1(TIMP- 1)基因对小鼠成纤维细胞生物学行为的影响。方法 应用正、反义人金属蛋白酶组织抑制因子 - 1(TIMP- 1)逆转录病毒表达载体转染 NIH3T3细胞 ,观察其对 NIH3T3细胞增殖的影响 ,Northern blot检测细胞 胶原 (α1)、MMP- 2、MMP- 9、TIMP- 1m RNA的表达 ,酶谱法分析细胞培养液中 MMP- 2、MMP- 9的蛋白表达 ;EL ISA测定细胞培养液中 FN、 、 、 型胶原的含量。结果 正义转染组显示出促细胞生长作用 (P<0 .0 1) ;反义转染组显示抑制细胞生长作用 (P<0 .0 1) ;TIMP- 1m RNA在各组均有表达 ,反义转染组表达明显减弱。正义转染组细胞培养液中 FN及 、 、 型胶原含量明显增高 (P<0 .0 1) ,MMP- 2 ,MMP- 9含量降低 ;而反义转染组 FN及 、 、 型胶原含量明显降低 (P<0 .0 1) ,MMP- 2 ,MMP- 9含量增高。结论 正义 TIMP- 1促进NIH3T3细胞的增殖 ,反义 TIMP- 1则抑制 NIH3T3细胞的增殖 ;反义 TIMP- 1通过抑制细胞 TIMP- 1的表达而促进小鼠成纤维细胞的 FN及 、 、 型胶原降解。

ObjectiveTo investigate the possible effects of tissue inhibitor of metalloproteinase-1(TIMP-1) on cell proliferation,matrix metalloproteinases(MMPs) and extracellular matrix(ECM) degradation in fibroblast.MethodsNIM3T3 cells transfected with recombinant mammalian expression retroviral vectors(pLXIN) carried sense human TIMP-1 cDNA(PTs) and antisense human TIMP-1 cDNA(PTas).Cell proliferation was measured by cell count.Type Ⅰ,Ⅲ and Ⅳ collagens and fibronectin(FN) content were tested in Cell culture supernatants by ELISA and MMP-2,MMP-9 content by Gelatin zymogram.Collagen-alpha 1 (Ⅳ) mRNA,MMP-2 mRNA,MMP-9 mRNA and TIMP-1 mRNA were analyzed by northern blot.ResultsProliferation of cultured NIH3T3 cells was enhanced in PTs transfected and was reduced in PTas transfected.A markedly decrease in expression of endogenous TIMP-1 mRNA in PTas transfected was confirmed by northern blot analysis.However,expressions of mRNA for collagen-alpha 1(Ⅳ),MMP-2,MMP-9 were not markedly changed.The concentrations of type Ⅰ,Ⅲ,Ⅳ collagnes and FN were markedly decreased in PTas transfected cells culture supernatants and markedly increased in PTs transfected cells culture supernatants.The contents of MMP-2,MMP-9,were markedly increased in PTas transfected cells culture supernatants and markedly decreased in PTs transfected cells culture supernatants.Conclusion These results suggested that antisense TIMP-1 mRNA suppress cell proliferation and the expression of endogenous TIMP-1,resulting in increase of ECM degradative activity of MMPs,hence it contributed to the degradation of ECM.