贵州医科大学学报

2015, v.40;No.172(01) 6-9

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细胞培养密度对MCF7乳腺癌细胞integrin β4水解的影响
The Proteolysis of Integrin β4 in MCF7 Breast Cancer Cell is Affected by Cell Density in Culture

严莉莎;霍建云;董宇华;潘娅;王晋星一;陈妮;臧贵勇;胡晓霞;陈腾祥;
YAN Lisha;HE Jianyun;DONG Yuhua;PAN Ya;WANG Jingxingyi;CHEN Ni;ZANG Guiyong;HU Xiaoxia;CHEN Tengxiang;Department of Physiology,Guiyang Medical College;Functional Laboratory,Guiyang Medical College;Department of Anatomy,Guiyang Medical College;

摘要(Abstract):

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 it's mechanism of cell density in culture on the proteolysis 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 localization 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 k D integrin β4 fragment was increased,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- 5mol / L,which was less than BAPTA-AM even if heparin reached to10- 4mol / L. Conclusions: In MCF7 cells with high density,calcium ion release from ion bank inside and inflow from outside of cells,leading to activation of calpain 2 and increase of product of integrin β4 fragment with 200 k D.

关键词(KeyWords): 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

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 严莉莎;霍建云;董宇华;潘娅;王晋星一;陈妮;臧贵勇;胡晓霞;陈腾祥;
YAN Lisha;HE Jianyun;DONG Yuhua;PAN Ya;WANG Jingxingyi;CHEN Ni;ZANG Guiyong;HU Xiaoxia;CHEN Tengxiang;Department of Physiology,Guiyang Medical College;Functional Laboratory,Guiyang Medical College;Department of Anatomy,Guiyang Medical College;

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DOI: 10.19367/j.cnki.1000-2707.2015.01.002

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