MMP-1过表达抑制人肝星状细胞I型胶原表达

Overexpression of MMP-1 significantly degrades type I collagen in human hepatic stellate cells

目的观察人肝星状细胞(LX-2)过表达基质金属蛋白酶-1(MMP-1)后,肝纤维化相关指标的变化。方法构建含MMP-1全长编码基因的表达质粒,并转染人肝星状细胞,荧光定量PCR与Western blot法分别检测MMP-1、I型胶原(collagenI)、TIMP-1基因与蛋白水平的表达。结果成功构建含MMP-1全长编码基因的表达质粒dl6-95-MMP-1;dl6-95-MMP-1转染LX-2细胞7d后,荧光定量PCR结果显示,MMP-1基因转录水平显著增加,而CollagenI、TIMP-1在基因转录水平无明显改变;Western blot结果显示转染7d后,MMP-1蛋白表达明显增加,CollagenI蛋白表达明显下降,而TIMP-1蛋白表达无明显变化。结论人肝星状细胞高表达MMP-1后显著抑制CollagenI蛋白的表达,其机制主要通过发挥其酶活性降解collagenI蛋白,而不影响collagenI基因水平的表达,同时也不影响TIMP-1在基因以及蛋白水平的表达。

Objective To construct recombinant vector carrying interstitial collagenase （MMP-1） and investigate whether the expression of collagen Ⅰ and other extracellular matrix ingredients could be inhibited in human hepatic stellate cell （LX-2） by overexpressing MMP-1 in vitro. Methods The plasmid expressing MMP-1 （d16-95-MMPI） was constructed through inserting the full length of MMP-1 gene into the AAV vector d16-95 which includes the ITR of AAV. Then, d16-95-MMP1 or d16-95 plasmid was transfected into LX-2 after 7 days. The levels of MMP-1, collagen I and TIMP-1 mRNAs and proteins were analyzed by real time PCR and Western blotting. Results The plasmid expressing MMP-1 （d16-95-MMP-1） was constructed successfully. After d16-95-MMP-1 plasmid was transfected into LX-2, real time PCR results showed that the level of MMP-1 mRNA increased significantly compared with LX-2 transfected with d16-95 plasmid （without MMP-1）, whereas the transcription of collagen I and TIMP-1 mRNA had not changed. Western blot results showed that MMP-1 increased significantly. After transfected with d16-95-MMP-1, the expression of collagen I was inhibited significantly, while the expression of TIMP-1 was not significantly changed. Conclusion MMP-1 overexpression in LX-2 could inhibit the expression of collagen Ⅰ significantly. This inhibition was mainly through degrading collagen I protein, other than through down-regulating the transcription of collagen Ⅰ and TIMP-1 mRNA or inhibiting the expression of TIMP-Ⅰ protein.