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目的:探讨整合素αVβ3在细胞外基质调控人黑色素瘤细胞基质金属蛋白酶2(matrixmetalloproteinase-2,MMP-2)表达和活化中的作用和地位。方法:选择黑色素瘤细胞系M21及突变型M21-L(M21-L不表达整合素αVβ3),通过观察2个细胞系在同一试验条件下的差异,研究整合素αVβ3在黑色素瘤细胞系M21与M21-L及其在MMP-2表达和活化中的作用;应用酶谱分析法检测黑色素瘤细胞系M21与M21-L,在包被I型胶原(type I collagen,Icol)、纤维黏连蛋白(fibronectin,FN)及ECM胶3种细胞外基质成分后,黑色素瘤细胞系M21与M21-L两者MMP?2表达量及活性的变化;通过RT-PCR方法检测包被后黑色素瘤细胞系M21与M21-L两者MT1-MMP mRNA表达水平的变化;通过Boyden小室膜侵袭实验观察黑色素瘤细胞系M21和M21-L细胞侵袭性差异。结果:培养于三维聚合Ⅰ型胶原、纤维黏连蛋白及ECM胶的M21细胞酶谱分析结果中出现了62 000的MMP-2的活化带,其膜型基质金属蛋白酶1(membrane?typematrixmetalloproteinase-1,MT1-MMP)mRNA表达均较没有包被3种细胞外基质的空白对照组增高;而M21-L在包被细胞外基质成分前后未出现MMP-2的活化,其MT1-MMP mRNA表达水平变化无统计学意义;Boyden小室检测表明,M21细胞侵袭力明显高于M21-L细胞。结论:整合素αVβ3参与了人黑色素瘤细胞M21的MMP-2表达和活化过程,是细胞外基质成分调控黑色素瘤细胞MMP-2表达和活化的必要条件。
Objective: To investigate the role and location of integrin αVβ3 in the regulation of matrix metalloproteinase-2 (MMP-2) expression and activation in human melanoma cells by extracellular matrix. Methods: The melanoma cell line M21 and mutant M21-L (M21-L do not express integrin αVβ3) were selected to observe the difference of the two cell lines under the same experimental conditions to investigate the effect of integrin αVβ3 on melanoma cell line M21 and M21-L and its role in the expression and activation of MMP-2. The melanoma cell lines M21 and M21-L were detected by zymography. After being coated with type I collagen (Icol), fibronectin (MMP-2) in the melanoma cell lines M21 and M21-L were detected by RT-PCR after the three extracellular matrix components of fibronectin (FN) and ECM glue were detected. The melanoma cell lines M21 and M21-L both MT1-MMP mRNA expression changes; Boyden chamber invasion assay observed melanoma cell lines M21 and M21-L cell invasive differences. Results: 62 000 MMP-2 activation bands were found in the enzyme-linked immunosorbent assay (MMA) of three-dimensional aggregated type I collagen, fibronectin and ECM glue. The membrane-type matrix metalloproteinase-1 , MT1-MMP mRNA expression was higher than that of the blank control group which was not coated with three kinds of extracellular matrix. However, M21-L did not show activation of MMP-2 before and after the coating of extracellular matrix. The expression level of MT1-MMP mRNA No statistically significant changes; Boyden chamber test showed that the invasiveness of M21 cells was significantly higher than M21-L cells. CONCLUSION: Integrin αVβ3 is involved in the process of MMP-2 expression and activation in human melanoma cell line M21 and is a necessary condition for the regulation of MMP-2 expression and activation in melanoma cells by extracellular matrix components.