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目的 :探讨正常乳腺上皮细胞周期蛋白E保持高分子量 (HMW )的机制。方法 :以乳腺肿瘤细胞为对照 ,采用蛋白质印迹法分析正常乳腺培养细胞裂解液经Ca2 + 处理后周期蛋白E分子量的变化 ,观察纯化的外源性钙离子激活中性蛋白酶 (CANP)对周期蛋白E的作用 ,同时观察和分析CANP小亚基在Ca2 + 作用下自我水解的程度。结果 :(1)正常乳腺细胞周期蛋白E主要以HMW形式表达 (分子量约 5 0kDa) ,而乳腺肿瘤细胞则表达大量 4 5kDa～ 35kDa小分子量 (LMW)周期蛋白E ;(2 )正常乳腺细胞匀浆经Ca2 + 处理后仅有很少量高分子量蛋白E水解为小分子片断 ,同时CANP小亚基也呈低水平水解 ;(3)预先用Ca2 + 激活的外源性CANP能使正常细胞HMW蛋白E全部转变成LMW蛋白 ,并伴随小亚基大量小分子水解片断的形成 ,而其内源性CANP小亚基仍保持原有分子量 ;(4 )Ca2 + 对正常乳腺细胞CANP活性产生负反馈式抑制效应。结论 :正常乳腺细胞周期蛋白E保持HMW形式与细胞内CANP活性保持稳态有关。
Objective: To investigate the mechanism of high molecular weight (HMW) of cyclin E in normal mammary epithelial cells. METHODS: Using mammary tumor cells as a control, the molecular weight of cyclin E after treatment with Ca2 + in lysates of normal breast culture cells was analyzed by Western blotting. The purified exogenous calcium-activated neutral proteinase (CANP) was observed for cyclins. The role of E, while observing and analyzing the degree of self-hydrolysis of the CANP small subunit under Ca2+ action. RESULTS: (1) Normal mammary cell cyclin E was mainly expressed in HMW form (molecular weight about 50 kDa), whereas mammary tumor cells expressed a large number of small molecular weight (LMW) cyclin E of 45kDa to 35kDa; (2) normal mammary gland cell After Ca2+ treatment, only a very small amount of high-molecular-weight protein E was hydrolyzed into a small molecule fragment, and the small subunit of CANP was also hydrolyzed at a low level; (3) Exogenous CANP activated with Ca2 + in advance could make normal cells HMW Protein E was completely converted to LMW protein, with the formation of a large number of small molecule hydrolyzed fragments of small subunits, while its endogenous small CANP subunit retained its original molecular weight; (4) Ca2 + negative feedback on normal breast cell CANP activity. The inhibitory effect. Conclusion : The retention of HMW forms in normal breast cyclin E is related to the steady state of intracellular CANP activity.