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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 study the mechanism of cell culture density on the mechanism of integrinβ4 in MCF7 breast cancer cells. Methods: High-density and low-density MCF7 breast cancer cells were cultured routinely. The cells were treated with calcium chelator and heparin respectively. Western blot was used to detect the expression of integrinβ4 in MCF7 cells. Fluo-3 AM was used as Ca2 + Confocal laser scanning microscopy to detect intracellular calcium distribution. Results: The results of Western blot showed that the mode of integrinβ4 hydrolysis was different in low and high density MCF7 cells; in high concentration cells, the increase of 200 kD integrinβ4 was observed. Confocal laser scanning microscopy In low-density culture of MCF7 cells, the green fluorescence of calcium ions mainly distributed in clusters or granules, and more red fluorescence overlap, part of the calcium ions distributed in the nucleus; in high-density cultured cells, calcium ions in the cytoplasm Membrane and nucleus were less localized. Calcium ion chelator BAPTA-AM significantly decreased the number of 200 kD integrinβ4, while 10-6 mol / L heparin decreased the number of 200 kD hydrolyzate in MCF7 cells. With the increase of heparin concentration Increased, the inhibitory effect has been enhanced; relative to BAPTA-AM, 10-4mol / L heparin inhibition decreased. CONCLUSION: In high-density cultured MCF7 cells, calcium is released from the calcium pool and extracellular influx increases. Activation of calpain 2 results in an increase of the 200 kD integrinβ4 hydrolyzed fragment.