Legumain对肿瘤细胞侵润力和血管内皮细胞管腔形成的作用  

Role of Legumain on Tumour Invasion and Human Endothelial Cells Tube Formation

作　　者：吴文苑 余涟 刘春 罗金刚[2] 段朝晖[2] 

机构地区：[1]深圳市人民医院检验科,深圳518020 [2]中山大学附属第二医院检验科,广州510120

出　　处：《热带医学杂志》2009年第8期864-867,F0004,共5页Journal of Tropical Medicine

基　　金：广东省自然科学基金(No.8151008901000035);深圳市医学科研基金(No.200802102)

摘　　要：目的探讨肿瘤组织中过度表达的一种天冬酰胺肽链内切酶Legumain在肿瘤细胞侵润性生长和血管内皮导管形成中的作用及相关机制。方法采用体外细胞培养的方法,观察在缺氧条件下人乳腺癌细胞MDA MB231和人静脉血管内皮细胞HUVEC中Legumain的蛋白表达量;观察Legumain对MDA MB231细胞的侵润能力的影响以及人工合成的Legumain抑制物(asparaginly endopepidase inhibitor,AEPI)对人静脉血管内皮细胞HUVEC小管腔形成能力的影响;用免疫组织化学的方法观察Legumain与整合素(Integrin)β1的结合情况。结果Western-blot的结果表明,在缺氧条件下,体外培养的人乳腺癌细胞MDA MB231和人静脉血管内皮细胞HUVEC的Legumain蛋白表达量增加。应用免疫荧光双染色法对缺氧条件下培养的MDA MB231细胞进行染色,与正常条件下培养的细胞相比,缺氧条件下培养的MDA MB231细胞体积增大,胞浆内含有大量绿色的Legumain,成点状分布,并有部分Legumain分布在迁移中的细胞表面前缘,与红色的整合素β1重叠,呈现为黄色。在血管内皮细胞小管腔形成试验中,加入AEPI的实验组,HUVEC细胞的小管形成数量明显减少,分别为(13.2±0.85),(5.33±0.35),(0.57±0.23),与对照组(21.3±1.73)相比差异均具有统计学意义(P<0.001)。细胞的侵润能力试验结果表明,加入激活的Legumain后,实验组中的MDA MB231细胞的侵袭能力增高,分别为(170.90±59.27和(321.22±22.39),与对照组(67.56±7.58)相比差异具有统计学意义(P<0.05)。结论缺氧条件下,MDA MB231细胞株和HUVEC细胞株的Legumain表达增加,能促进肿瘤细胞的迁移和血管内皮细胞的小管形成。其分子机理之一可能是通过与整合素β1结合。Objective To investigate the role of Legumain, an asparaginyl endopeptidase overexpressed by tumor tissue, in tumor invasive growth and angiogenesis. Method Western-blot was use to study the expression of Legumain in cells under normal or hypoxic condition. The migration of MDA MB231 cells were detected by Transwell invasion assay. The tube formation by HUVEC was detected by tube formation test. The binding of Legumain to integrin was analyzed by immunohistochemical method. Result The expression of Legumain in both MDA MB231 and HUVEC were increased under hypoxia. Using fluorescence double staining method, hypoxic MDA MB231 cells were found to have an increased in size of the cell body. Legumain was found, as shown in green dots, inside of the ceils. Some of the Lugumain were found on the cell surfaces and overlapping with integrin （red） on the margin of the cell surfaces （yellow）. The formation of endothelial tube was reduced by Legumain inhibitor （an asparaginly endopepidase inhibitor, AEPI）. Compared with the control group （21.3±1.73）, the numbers of tube were （13.2±0.85）, （5.33±0.35） and （0.57±0.23）, （P〈0.001）. Results of cell migration assay showed that legumain stimulated the migration of MDA MB231 cells （170.90± 59.27 and 321.22±22.39）, when compared with the control group （67.56± 7.58,P〈0.05）. Conclusion Overexpression of Legumain was found to promote the migration of hypoxic MDA MB31 cells and tube formation by HUVECs. The effect might be due to the binding of legumain to integrinβ1.

关 键 词：肿瘤 LEGUMAIN HUVEC细胞株 MDA MB231细胞株 

分 类 号：R737.9[医药卫生—肿瘤]