细胞培养密度对MCF7细胞calpain2和FAK的水解及活性影响

目的：探讨细胞培养密度对乳腺癌MCF7细胞钙激活中性蛋白酶2（calpain 2）和焦点黏附蛋白（FAK）的水解及活性的影响.方法：常规培养乳腺癌细胞MCF7,分别以1×10^6、5×10^6、1×10^7个细胞传代于不同的6 cm皿中,培养36 h后,形成不同的培养密度（分别为低密度、正常密度和高密度）,裂解细胞,提取蛋白行Western blot检测calpain 2和FAK的表达,用磷酸化抗体检测FAK的Y397位点磷酸化水平;MCF7细胞高密度培养并calpain 2抑制剂处理后,用Western blot法检测FAK水解情况和磷酸化水平.结果：不同密度培养对calpain 2的表达没有明显影响,但可影响calpain 2的水解,培养密度加大,水解程度加强;培养密度也可以影响FAK的水解模式,低密度培养细胞的FAK以较大的水解片段为主,高密度培养的FAK则以较小片段为主,高密度培养细胞FAK Y397磷酸化水平比低密度的低;给予calpain 2特异性抑制剂后,高密度培养细胞FAK大片段增多,小片段减少,其Y397位点的磷酸化水平增高.结论：细胞高密度培养可使MCF7细胞calpain 2的活性增强,FAK的水解程度加大,降低FAK的磷酸化.

The Effects of Cell Density on the Proteolysis and Activity of Calpain 2 and FAK in MCF7 Cell

Objective： To explore the effects of cell density on the proteolysis and activity of calcium- activated neutral protease 2 （ ealpain 2 ） and focal adhesion kinase （FAK）. Methods： MCF7 breast cancer cells with normal culturing were planted into 6 cm dishes with different number to form varied density. The protein samples of MCF7 ceils with different density were extracted and loaded to SDS- PAGE, and western blot to detect the expression, proteolysis and phosphorylation of calpain 2 and FAK. Calpain 2 specific inhibitor, calpastatin and calpain inhibitor IV ,were used to treat MCF7 cell with high density and to evaluate relationship between calpain 2 and FAK. Results： The proteolysis of calpain 2, but not expression level, was affected with cell density, the higher density, the more prote- olysis. The proteolysis of FAK was affected with cell density too and consequently with the alteration of phosphorylation of Y397. More small fragments of FAK and less phosphorylation of Y397 were pro- duced in high density than those in low density, which were inversed by calpain 2 specific inhibitors. Conclusion： Calpain 2 of MCF7 cell can be activated in high density culture, leading to high level proteolysis and less Y397 phosphorylation of FAK.