摘要
目的肝纤维化的主要病理变化是细胞外基质的沉积与降解障碍,本研究旨在观察大鼠肝星状细胞过表达基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)后肝纤维化相关指标的变化。方法构建含MMP-13全长编码基因的表达质粒,并转染大鼠肝星状细胞(hepatic stellate cell,HSC),荧光定量PCR与Western blotting方法分别检测MMP-13、CollagenI基因与蛋白水平的表达。结果成功构建含MMP-13全长编码基因的表达质粒dl6-95-MMP-13;dl6-95-MMP-13转染大鼠HSC细胞7d后,荧光定量PCR结果显示,MMP-13基因转录水平明显增加,而CollagenI在基因转录水平有轻度增加;Western blotting结果显示转染7d后,酶原及活化形式的MMP-13表达明显增加,尤其是活化形式的MMP-13;转染后的大鼠HSC中CollagenI蛋白表达明显下降。结论大鼠HSC高表达MMP-13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,而对CollagenI在基因水平的表达无显著影响。
Objective Liver fibrosis is characterized by increased deposition and altered composition of extracellular matrix. The aim of this study was to construct recombinant vector carrying interstitial collagenase （MMP-13） and investigate if the expression of collagen I could be inhibited in rat hepatic stellate cell（HSC） overexpressing MMP-13 in vitro. Methods The plasmid expressing MMP- 13 （dl6-95-MMP-13） was constructed through inserting the full length of MMP-13 gene into the AAV vector dl6-95 which includes the ITR of AAV. Then, dl6-95-MMP-13 or dl6-95 plasmid was transfected into HSC for 7 days. The level of MMP-13 and collagen I mRNAs and proteins were analyzed by real time PCR and Western blotting. Results The plasmid expressing MMP-13 （dl6-95-MMP-13） was constructed successfully. After dl6-95-MMP-13 plasmid was transfected into HSC for 7 days, real time PCR results showed that the level of MMP-13 mRNA increased significantly compared with HSC transfeeted with dl6-95 plasmid （without MMP-13 ）, whereas the transcription of collagen I had only slight increase. Western blotting results showed that both the pro-MMP-13 and active MMP-13 increased significantly, especially the active form of MMP-13. After transfeeted with dl6-95-MMP-13, the expression of collagen I was inhibited significantly. Conclusion MMP-13 overexpression in HSC could inhibit the expression of collagen I significantly. This inhibition occurred mainly through degradation of collagen I protein, rather than through regulating the transcription of collagen I.
出处
《首都医科大学学报》
CAS
北大核心
2009年第3期313-316,共4页
Journal of Capital Medical University
基金
国家高技术研究发展计划(2006AA02A410)
国家自然科学基金(30500425)资助项目~~
