外源性组织金属蛋白酶抑制剂-1对基质金属蛋白酶-9高表达大鼠皮肤成纤维细胞生物学行为的影响  

Effect of tissue inhibitor of metalloproteinase 1 on the biological behaviors of rat dermal fibroblast with high matrix metalloproteinase 9

作　　者：薛声能[1] 雷娟[2] 杨川[1] 林刁珠[1] 朱平[1] 徐明彤[1] 严励[1] 

机构地区：[1]广州中山大学孙逸仙纪念医院内分泌科,510120 [2]广州中山大学孙逸仙纪念医院心血管科,510120

出　　处：《中华糖尿病杂志》2012年第2期115-119,共5页CHINESE JOURNAL OF DIABETES MELLITUS

基　　金：国家自然科学基金项目（81070660）;广东省科技计划基金项目（2008A030201012,20098091300128）

摘　　要：目的观察外源性组织金属蛋白酶抑制剂-1（TIMP-1）对基质金属蛋白酶-9（MMP-9）高表达大鼠皮肤成纤维细胞生物学行为的影响，为寻找治疗糖尿病足的新方法提供实验依据。方法应用sD大鼠皮肤成纤维细胞株CRL-1213用高糖（22mmol／L）＋高同型半胱氨酸（100μmol／L）联合培养建立体外MMP-9高表达皮肤成纤维细胞模型，用外源性TIMP-1（100μg／iJ）抑制MMP-9的活性，采用实时PCR法和ELISA法检测MMP．9基因及蛋白表达量，用明胶酶谱法检测MMP-9蛋白活性，采用流式细胞术检测细胞增殖功能、CCK-8检测细胞活力、ELISA法检测细胞胶原（羟脯氨酸）分泌能力、划痕实验评价细胞横向迁移能力、Transwell法评价细胞纵向迁移能力。采用单因素方差分析进行统计学分析。结果TIMP-1干预组大鼠皮肤成纤维细胞MMP-9mRNA和蛋白的表达量与MMP-9高表达组比较差异无统计学意义（P〉0．05），但MMP-9蛋白活性受到明显抑制（1．47±0．13VS0．434-0．13，t=12．495，P〈0．01）。与对照组比较，MMP-9高表达组大鼠皮肤成纤维细胞S期的比例[（9．31±0．24）％VS（6．54±0．29）％]、增殖指数[（13．8±0．5）％VS（11．3±0．6）％]、细胞活力（1．76±0．13V81．35±0．12）、胶原（羟脯氨酸）分泌量[（1126-1-63）vs（353±61）μg／L]、6h横向迁移率[（38．7±2．6）％VS（21．3±2．1）％]和纵向迁移细胞数（91±4vs71±4）均明显减少（t=16．433、7．328、5．113、19．847、11．529、8．124，均P〈0．05）；TIMP-1干预后，大鼠皮肤成纤维细胞S期的比例[（6．54±0．29）％VS（7．75±0．27）％]、增殖指数[（11．3±0．6）％vs（12．5±0．5）％]、细胞活力（1．35±0．12VS1．64±0．14）、胶原（羟脯氨酸）分泌量[（3534-61）vs（829±59）仙g／L]、6h横向迁移率[（21．3±2．1）％VS（31．5±2．7）％]和纵向迁移细胞数（71±4vs85±4）与MMPObjective To explore the effect of tissue inhibitor of metalloproteinase 1 （TIMP-1） on the biological behaviors of rat dermal fibroblast with high matrix metalloproteinase 9 （ MMP-9）, in order to provide an experimental basis for looking for a new way to treat diabetic foot. Methods The cell model of skin fibroblast was established by using dermal fibroblast cell line CRL-1213 from SD rats with high expression of MMP-9 by high glucose （22. 0 mmol/L） ＋ high homocysteine （ 100 μmol/L） co-culture. Exogenous TIMP-1 was used to inhibite the activity of MMP-9. Realtime PCR and ELISA were used to detect the expression of MMP-9 mRNA and protein. Gelatin zymography was used to detect the activity of MMP-9.Flow cytometry was used to detect cell proliferation. CCK-8 was used to detect cell viability. ELISA assay was used to detect collagen （hydroxyproline） secretion. Scratch test was used to evaluate horizontal migration of cells. Transwell method was used to evaluate vertical migration of cells. ANOVA was used for data analysis. Results The expression difference of MMP-9 mRNA and protein between TIMP-1 group and high MMP-9 group had no statistically significant （P 〉 0. 05）. But the activity of MMP-9 protein in TIMP-1 group was lower than that in high MMP-9 group （ 1.47 ± 0. 13 vs 0. 43 ＋ 0. 13, t = 12. 495, P 〈 0. 01 ）.group was lower than that in high MMP-9 group （ 1.47 ±0. 13 vs 0. 43 ＋ 0. 13, t = 12. 495, P 〈 0. 01 ）. Compared with control group, the proportion of S phase cells [ （ 9. 31 ± 0. 24 ） % vs （ 6. 54 ± 0. 29 ） % ], proliferation index [ （13.8 -＋ O. 5 ）% vs （ 11.3 ~ 0.6）% ], cell viability （ 1.76 ±0. 13 vs 1.35 ±0. 12 ）, collagen （hydroxyproline） secretion [ （ 1126± 63 ） vs （353 -＋ 61 ） μg/L ], 6 h horizontal migration rate [（38.7-＋2.6）% vs （21.3 ＋2.1）%± and the number of vertical migration cells （91＋4 vs71 _＋4） in high MMP-9 group were decreased （t = 16. 433,7. 328,5. 113,19. 847,11. 529 and 8. 124 r

关 键 词：基质金属蛋白酶9 组织金属蛋白酶抑制剂1 糖尿病足 大鼠 

分 类 号：R285.5[医药卫生—中药学]