摘要
目的:研究细胞培养密度对MCF7乳腺癌细胞integrinβ4水解机制。方法:常规培养高密度和低密度MCF7乳腺癌细胞,分别给予钙离子螯合剂和肝素处理,用Western blot检测不同培养密度MCF7细胞integrinβ4的水解情况;用Fluo-3 AM作为钙离子荧光探针,激光共聚焦扫描显微镜检测细胞内钙离子的分布。结果:Western blot检测结果显示,在低密度和高密度培养的MCF7细胞中,integrinβ4的水解模式不同;在高浓度培养的细胞中,200 k D integrinβ4片段产生增多;激光共聚焦扫描显微镜观察发现,低密度培养MCF7细胞中,钙离子的绿色荧光主要成团或颗粒状分布,而且与红色荧光有较多的重合,部分钙离子分布于细胞核内;在高密度培养的细胞中,钙离子在细胞质膜和细胞核中的定位较少;钙离子螯合剂BAPTA-AM处理可使200 k D integrinβ4明显减少;10-6mol/L的肝素能够减少MCF7细胞200 k D水解片段的增多,随着肝素浓度的增高,抑制效果有所增强;相对于BAPTA-AM,10-4mol/L肝素的抑制效应减弱。结论:高密度培养的MCF7细胞中,钙离子从钙库中释放,细胞外内流增多,激活calpain 2,导致200 k D integrinβ4水解片段增多。
Objective： To investigate the role and itg mechanism of cell density in culture on the pro- teolysis of integrin β4. Methods： MCF7 was cultured to reach 70% （sparse） and 100% （dense） and then treated with calcium ion chelator and heparin. Western blot was utilized to detected the proteolysis of integrin β4, and Fluo-3 AM was used as fluorescence probe for calcium ion to observe its localiza- tion inside cell. Results： As Western blot results showing, the proteolysis profile of integrin β4 was different between cells cultured as sparse and dense. In dense, 200 kD integrin β4 fragment was in- creased, which could be attenuated by BAPTA-AM （calcium ion chelator） and heparin. However, the inhibitor effects of heparin was dose-dependent, but reached to its platform as 10-5 mol/L, which was less than BAPTA-AM even if heparin reached to10-4 mol/L. Conclusions： In MCF7 ceils with high density, calcium ion release from ion bank inside and inflow h＇om outside of cells, leading to activation of calpain 2 and increase of product of integrin β4 fragment with 200 kD.
出处
《贵阳医学院学报》
CAS
2015年第1期6-9,共4页
Journal of Guiyang Medical College
基金
国家自然科学基金资助项目(NO.81060176)
贵阳医学院院基金(院基金合同字第2012005号)
贵州省高层次人才科研条件特助经费(TZJF-2009年38号)
关键词
钙离子
培养密度
整合素β4
钙激活中性蛋白酶2
肝素
calcium ion
density of cell culture
integrein β4
calcium-activated neutral proteases 2
heparin
