反义TIMP_1基因转染Ito细胞后对其ECM代谢的影响  

Effects of antisense TIMP1 gene on extracellular matrix metabolism in Ito cells in vitro

作　　者：曾仲[1] 韩本立[1] 

机构地区：[1]第三军医大学附属西南医院全军肝胆外科研究所,西南肝胆外科医院,重庆400038

出　　处：《第三军医大学学报》2002年第6期709-712,共4页Journal of Third Military Medical University

摘　　要：目的　探讨反义TIMP1基因对Ito细胞 (贮脂细胞 )金属蛋白酶的表达和细胞外基质 (ECM )降解的影响。方法　将大鼠全长TIMP1cDNA反向插入哺乳表达载体pCI neo中 ,构建反义TIMP1基因真核表达载体 ,用脂质体介导的方法 ,将反义TIMP1基因导入Ito细胞株CFSC 2G中 ,选择稳定转染的Ito细胞株培养 ,采用RT PCR、原位杂交、免疫组化及RIA等方法 ,观察TIMP1mRNA和蛋白、MMP1mRNA和蛋白、α1(Ⅲ )mRNA、FN蛋白表达及细胞培养上清中PCI、PCⅢ、HA、LN水平的变化。结果　反义TIMP1基因能明显地抑制Ito细胞中TIMP1mRNA和蛋白的高表达 (P <0 0 1)。转基因细胞中α1(Ⅲ )mRNA、MMP1mRNA、FN蛋白的表达与未转基因细胞比较无明显变化 (P >0 0 5 )。转基因后Ito细胞培养上清中PCI、PCⅢ、HA、LN水平明显低于未转基因细胞 (P <0 0 1)。结论　反义TIMP1基因可以抑制贮脂细胞内源性TIMP1产生 ,但未影响MMP1的表达 。Objective To investigate the possible role of antisense TIMP1 gene on expression of MMP and the extracellular matrix metabolism of Ito cells. Methods A rat tissue inhibitor of metalloproteinase 1 (TIMP1) cDNA (750 bp) was inserted reversely into the mammalian expression vector pCI neo. The recombinant antisense TIMP1 gene mammalian expression vector pCIATIMP1 was transfected into rat Ito cells line CFSC 2G with a liposome mediated technique. By using RT PCR, ISH and immunohistochemistry, the expression of TIMP1 mRNA and protein, MMP1 mRNA and protein,α1(Ⅲ) mRNA and FN protein were determined. The concentrations of PCI, PCⅢ, HA and LN in the Ito cells culture media were determined with RIA. Results Antisense TIMP1 gene were markedly suppressed the expression of endogenous TIMP1 mRNA and protein in the transfected Ito cells ( P <0.01). The expression of MMP1 mRNA and protein,α1(Ⅲ)mRNA and FN protein were not significantly different in the transfected Ito cells. The levels of PCI, PCⅢ, HA and LN were decreased significantly in the transfected Ito cells culture media ( P <0.01). Conclusion The antisense TIMP1 gene can be successfully used to inhibit Ito cell endogenous TIMP1 mRNA and protein, have no effect on the expression of MMP1, and enhance the degradation of extracellular matrix.

关 键 词：反义TIMP1基因 ITO细胞 细胞上基质 肝纤维化 

分 类 号：R575.2[医药卫生—消化系统] Q344[医药卫生—内科学]