化学缺氧对肝星状细胞基质金属蛋白酶-2表达及活性的影响  

Hypoxia induced by COCl2 influencing the expression and the activity of matrix metalloproteinase-2 in rat hepatic stellate cells

作　　者：范任华[1] 陈平圣[1] 赵迪[1] 张万东 

机构地区：[1]东南大学基础医学院病理学教研室,南京210009 [2]加拿大国家研究院生物科学研究所

出　　处：《中华肝脏病杂志》2007年第9期654-657,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金（30470780）

摘　　要：目的探讨化学缺氧对大鼠HSC MMP-2的表达和活性的影响及其调节机制。方法用不同浓度的氯化钴（CoCl2,0、50、100、200μmol/L）造成大鼠HSC化学缺氧,RT-PCR、酶图法分别检测MMP-2 mRNA表达及其酶活性;Western blot检测缺氧诱导因子-1α（HIF-1α）表达;用荧光素酶报告基因分析技术测HIF-1α对MMP-2基因的激活作用。结果随CoCl2浓度从0μmol/L依次递增到200μmol/L,MMP-2 mRNA表达的量（用吸光度值表示）从0.53±0.12依次上调到1.57±0.11,4组间差异有统计学意义（F=34.21,P〈0.01）,其活性下降幅度（用吸光度值表示）从84.49±5.38逐渐下降至53.70±3.42,4组间差异有统计学意义（F=29.54,P〈0.01）,HIF-1α表达量依次增加;核蛋白提取物与MMP-2基因探针（含缺氧反应元件）共浴后,电泳迁移条带产生延迟现象,竞争性序列能部分拮抗其结合。结论化学缺氧能使HSC MMP- 2 mRNA表达上调,酶活性下降。HIF-1α可能参与了缺氧条件下MMP-2的表达调节。Objectives To investigate the effects of hypoxia induced by cobalt chloride on the expression and the activity of matrix metalloproteinase-2 （MMP-2） in rat hepatic stellate cells （HSC-T6） and to clarify the possible mechanisms. Methods HSC-T6 cell line was grown in Dulbecco＇s modified Eagle medium with 10% fetal calf serum at 370C and 5% CO2. When reaching confluence, the cells were incubated with serum-free medium in the presence of cobalt chloride （0, 50, 100, 200 μ mol/L） for six hours, and then the supernatant and the cells were harvested. The expression of the MMP-2 mRNA and HIF- 1 α protein in HSC-T6 cells was detected using RT-PCR and Western blot respectively. The activity of the MMP-2 in the supernatant was detected by zymography. The binding reaction between HIF- 1 α protein and MMP-2 gene sequence was investigated by electrophoresis mobility shift assay. Results When the concentration of COCl2 increased from 0 μ mol/L to 200 μ mol/L, the expressions of MMP-2 mRNA （the rate of light density） were increased from 0.53 ± 0.12 to 1.57 ± 0.11 and the differences among these four groups were significant （F = 34.21, P 〈 0.01 ）. The activity of MMP-2 （the value of light density x band area） decreased gradually from 84.49 ± 5.38 to 53.70 ± 3.42, and the differences among these four groups were also significant （F = 29.54, P 〈 0.01）. The expressions of HIF- 1α were increased gradually with the increase of the COCl2 concentration. The shift band in the lane of the nuclear protein extraction and the MMP-2 probe containing hypoxia response element showed delays when compared with the lane of the sole probe, and the binding was partially abolished when competing sense oligonucleotides were used. Conclusions Our results suggest that chemical hypoxia can up-regulate the expression of MMP-2 mRNA and decrease the activity of the enzyme. HIF-1 α may play a part in the regulation of MMP-2 transcription under hypoxic conditions.

关 键 词：肝纤维化 缺氧 基质金属蛋白酶-2 肝星状细胞 缺氧诱导因子-1Α 

分 类 号：R686[医药卫生—骨科学]