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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1×106、5×106、1×107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。
Objective: To investigate the effect of cell culture density on the hydrolysis and activity of calpain 2 and focal adhesion protein (FAK) in breast cancer MCF7 cells. Methods: Breast cancer cells MCF7 were routinely cultured and passaged in different 6 cm dishes with 1 × 106, 5 × 106 and 1 × 107 cells, respectively. After cultured for 36 h, different culture densities (low density, normal Density and high density). The cells were lysed and extracted for Western blot to detect the expression of calpain 2 and FAK. Phosphorylation antibody was used to detect the phosphorylation level of Y397 in FAK. After MCF7 cells were treated with calpain 2 inhibitor at high density, Western blot assay FAK hydrolysis and phosphorylation level. Results: Cultured at different densities had no significant effect on the expression of calpain 2, but could affect the hydrolysis of calpain 2, the culture density increased and the degree of hydrolysis strengthened. The culture density also affected the hydrolysis pattern of FAK, and the FAK of low density cultured cells was larger Of the FAK Y397 phosphorylation level was lower than that of the low density. The calpain 2-specific inhibitor was given to high-density cultured FAK large fragment Increased, a small fragment decreased, the phosphorylation of Y397 site increased. CONCLUSION: High-density cell culture can enhance the activity of calpain 2 in MCF7 cells, increase the degree of hydrolysis of FAK, and decrease the phosphorylation of FAK.