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目的 研究缺氧、高氧对肝星形细胞基质金属蛋白酶Ⅱ (MMP 2 )表达及其活性影响。方法 分离大鼠肝星形细胞 ,在缺氧或高氧条件下培养 ,以免疫细胞化学标记链霉素卵白素生物素(LSAB)法及酶联免疫吸附 (ELISA)法分别检测细胞内MMP 2、基质金属蛋白酶组织抑制因子Ⅱ(TIMP 2 )、膜型基质金属蛋白酶Ⅰ (MT1 MMP)的表达 ,和培养上清中MMP 2、TIMP 2的相对含量 ,并以酶谱法测培养上清MMP 2活力。结果 (1)缺氧培养 12h ,MMP 2表达增高 (缺氧组阳性指数 :5 7±2 0 ;对照组 :3 2± 1 0 ,;P <0 0 1) ,TIMP 2表达则降低 (缺氧组阳性指数 :2 5± 0 7;对照组 :3 6±1 0 ;P <0 0 5 ) ;培养上清中MMP 2酶活性明显降低 (缺氧组总吸光度值 :7 334± 1 92 2 ;对照组 :17 2 77± 7 4 2 4 ;P <0 0 1)。缺氧不同时段 (6、12、2 4h)比较 ,变化以 6h段最明显。 (2 )高氧培养 12h ,上清中MMP 2、TIMP 2蛋白相对含量均高于对照组 ,TIMP 2更明显 (高氧组A450 :0 0 5 0± 0 0 14 ;对照组 :0 0 2 2± 0 0 10 ;P <0 0 1) ,酶活性亦高于对照组 (高氧组总吸光度值 :5 2 5 2± 0 771;对照组 :4 30 4± 1 0 83;P <0 0 5 ) ,高氧组MT1 MMP表达增强。结论 肝星形细胞对氧敏感。缺氧使肝星形细胞MMP 2表达增加 ,该影响在?
Objective To study the effect of hypoxia and hyperoxia on the expression and activity of matrix metalloproteinase 2 (MMP 2) in hepatic stellate cells. Methods Rat hepatic stellate cells were isolated and cultured in hypoxia or hyperoxia condition. The levels of MMP2 (superscript +) and MMP2 were detected by immunocytochemical streptavidin biotin (LSAB) and enzyme - linked immunosorbent assay (ELISA) , The expression of tissue inhibitor of metalloproteinase (TIMP 2) and membrane-type matrix metalloproteinase-1 (MT1 MMP), and the relative content of MMP 2 and TIMP 2 in the culture supernatant, and the activity of supernatant MMP 2 vitality. Results (1) The expression of MMP-2 increased after hypoxia for 12 hours (positive index in hypoxia group: 57 ± 20; control group: 32 ± 10, P <0.01) The positive index of oxygen group: 25 ± 0 7; control group: 36 ± 1 0; P <0 05). The activity of MMP 2 in the culture supernatant was significantly decreased (total absorbance value in the hypoxia group: 7 334 ± 1 92 2; control group: 17 2 77 ± 7 4 2 4; P <0 0 1). Hypoxia different periods (6,12,2 4h), the change in 6h most obvious. (2) Compared with the control group, the relative content of MMP 2 and TIMP 2 in the supernatant of the supernatant was significantly increased after 12 h of hyperoxia incubation (P <0.05). TIMP 2 was more obvious in the hyperoxia group (A450: 0 0 5 0 ± 0 0 14 in the hyperoxia group and 0 0 2 2 ± 0 0 10; P 0 01), and the enzyme activity was also higher than that of the control group (the total absorbance value of the high oxygen group was 5252 ± 0 771; the control group was 4 30 4 ± 1 0 83; P <0 0 5), MT1 MMP expression increased in hyperoxia group. Conclusion Hepatic stellate cells are sensitive to oxygen. Hypoxia increases the expression of MMP-2 in hepatic stellate cells.