Transwell小室筛选高侵袭性C6细胞MMP-2和TIMP-2的表达

Expressions of MMP-2 and TIMP-2 in C6 cells with high invasive ability selectedthrough Transwell chambers

目的:通过观察Transwell小室筛选的高侵袭性C6细胞中组织金属蛋白酶抑制剂-2(tissueinhibitorofmetalloproteinase-2,TIMP-2)的表达,探讨基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)及TIMP-2在胶质瘤侵袭中的作用机制。方法:通过Transwell小室筛选出一种更具有侵袭能力的C6-C1细胞,用细胞损伤愈合实验及Transwell小室评价其侵袭转移能力,用明胶酶谱、反向明胶酶谱及Westernblot等方法来检测MMP-2和TIMP-2的表达。结果:与同源C6细胞相比,这种体外筛选的C6-C1侵袭能力增加3倍多,增强了快速的单层细胞损伤愈合能力,MMP-2激活水平增加80%,TIMP-2的蛋白表达水平增长2倍。结论:MMP-2的激活水平增加与胶质瘤的侵袭转移能力相关,并与TIMP-2的表达增高相关。TIMP-2表达增加促进了MMP-2的激活和细胞侵袭。

OBJECTIVE:To explore the roles of matrix-metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in glioma invasion and motility by detecting the expression of TIMP-2 in C6 cells selected through Transwell chambers. METHODS: Transwell chambers were used to select for a more potently invasive subpopulation of C6 cells. Both wound healing and Transwell invasion assay were used to assay the ability of the selected invasive C6 cells. Expressions of MMP-2 and TIMP-2 were detected by gelatin zymography, reverse zymography and Western blot. RESULTS: The stable population of tumors cells (C6-C_1) obtained through selection process in vitro were three times more invasive than parental C6 cells and demonstrated faster monolayer wound healing. The enhanced invasiveness is associated with an 80% increase in MMP-2 activation. However, C6-C_1 cells exhibited two-fold elevation of TIMP-2 protein expression relative to parental cells. CONCLUSIONS: The increased levels of MMP-2 activation is associated with the increased ability of glioma invasion and the increased levels of TIMP-2 expression. An increase in physiological levels of TIMP-2 can promote MMP-2 activation and invasion in glioma cells.