摘要
目的 探讨上皮型钙黏连蛋白（E-cadherin,E-cad）表达对MDA-MB-231人乳腺癌细胞黏附力及增殖的影响.方法 采用脂质体转染方法将携带有E-cad基因的重组真核表达质粒E-cadpcDNA3转染入E-cad阴性MDA-MB-231人乳腺癌细胞系,通过G418筛选,Western blot法鉴定出E-cad过表达的阳性单克隆细胞株.分别通过三种不同的黏附试验检测E-cad转染前后细胞自身黏附、与基质黏附及与其他细胞黏附能力的变化：免疫沉淀法检测表达的外源性E-cad能否与内源性连环蛋白（β-catenin,β-cat）发生相互作用.Western blot检测E-cad表达后细胞内β-cat、细胞周期蛋白（cyclin）D1蛋白的表达变化;MTT法检测细胞的生长和增殖能力状况;流式细胞仪检测转染E-cad前后细胞凋亡变化情况.免疫细胞化学直接二步法检测β-cat定位变化.结果 通过稳定转染,获得了稳定表达E-cad的两个细胞株Ecad-231-7和Ecad-231-9.MDA-MB-231细胞在不贴壁培养时大部分呈单细胞状态,而两个表达E-cad的细胞株都形成大细胞团;MDA-MB-231、Ecad-231-7和Ecad231-9与HCT116细胞共孵育30 min后的平均黏附率分别为39.0%、60.0%和59.5%.MDA-MB-231细胞在EDTA作用5 min后的平均脱落率为37.4%,Ecad-231-7和Ecad-231-9细胞分别下降为4.2%和7.2%,即E-cad表达后乳腺癌细胞的自身黏附力、与基质的黏附力及与其他细胞的黏附力均增强;且细胞内过表达的外源性的E-cad与β-cat结合,使细胞内cyclin D1表达下降,细胞增殖受到抑制.常规培养48 h后MDA-MB-231、Ecad-231-7和Ecad-231-9细胞的凋亡率分别为1.9%、2.0%和2.1%,E-cad表达对细胞的凋亡没有明显影响.二步法染色显示胞质内β-cat增加.结论 成功建立了稳定表达E-cad蛋白的单克隆细胞株Ecad-231-7和Ecad-231-9.表达的E-cad通过β-cat-cyclinD1途径增强癌细胞黏附力、抑制癌细胞增殖.
Objective To investigate the role that E-cadherin （E-cad） plays on cell adhesion and proliferation of human breast carcinoma. Methods E-cad expression vector was transfected into an E-cadnegative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad,β-catenin （β-cat） and cyclin DI expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/β-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of β-cat. Result E-cad （ ＋ ） cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4. 2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous β-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2. 0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture.β-cat increased in the cytoplasma. Conclusions Two monoclonal tumor cell strains （ Ecad-231-7 and Ecad-231-9） stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving β-cat and cyclin D1.
出处
《中华病理学杂志》
CAS
CSCD
北大核心
2010年第12期842-847,共6页
Chinese Journal of Pathology
