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目的筛选不同转移潜能的乳腺癌细胞亚系,并探讨皮质肌动蛋白(cortactin)在乳腺癌细胞增殖和侵袭过程中的作用。方法经过连续人工基质膜侵袭实验后获得高、低转移潜能的乳腺癌细胞亚系,通过透射电镜观察比较两系细胞的超微结构,四甲基偶氮唑盐(MTT)法检测两系细胞增殖能力,流式细胞仪检测两系细胞周期,Transwell侵袭小室模型比较两系的迁移能力。应用免疫细胞化学法、反转录聚合酶链反应(RT-PCR)和蛋白质印迹分析法检测cortactin蛋白在两系细胞中的表达。结果利用人工基质膜侵袭实验筛选出高、低转移潜能乳腺癌细胞亚系,并且两系细胞形态上无明显差异。MTT实验显示高转移潜能细胞体外生长速度显著快于低转移潜能细胞(P<0.05)。流式细胞仪检测结果显示,高转移潜能细胞亚系与低转移潜能细胞亚系相比,G0/G1期细胞前者比后者少[(52.67±3.69)%vs(64.46±2.79)%],而S期细胞前者比后者多[(30.53±6.19)%vs(24.63±2.04)%]。高转移潜能细胞亚系增殖指数(PI)高于低转移细胞亚系[(47.32±3.69)%vs(35.53±2.80)%,P<0.05],侵袭能力显著强于低转移潜能细胞亚系[(61.46±7.08)vs(25.32±4.87)个/视野,P<0.05]。免疫组化、RT-PCR和蛋白质印迹分析均显示在高转移潜能细胞亚系中cortactin在基因和蛋白水平的表达均高于低转移潜能细胞亚系(P<0.05)。结论 Cortactin的过表达与乳腺癌的增殖和转移过程密切相关。
Objective To screen breast cancer cell lines with different metastatic potentials and to explore the role of cortactin in the proliferation and invasion of breast cancer cells. Methods The breast cancer cell lines with high and low metastatic potential were obtained after continuous artificial matrix membrane invasion test. The ultrastructures of the two lines of cells were observed by transmission electron microscopy. The two lines of cells were detected by MTT assay. Proliferative ability, flow cytometry two-line cell cycle detection, Transwell invasion chamber model compared two lines of migration ability. Immunocytochemistry, RT-PCR and Western blotting were used to detect the expression of cortactin protein in the two lines of cells. Results High and low metastatic potential breast cancer cell lines were screened by artificial matrix membrane invasion assay. There was no significant difference in morphology between the two lines. MTT experiments showed that the high metastatic potential cells in vitro grew significantly faster than the low metastatic potential cells (P <0.05). The results of flow cytometry showed that the cells in G0 / G1 phase were less (52.67 ± 3.69)% vs (64.46 ± 2.79)% than those in the low metastatic potential cell subline) While the former S cells were more than the latter [(30.53 ± 6.19)% vs (24.63 ± 2.04)%]. The proliferation index (PI) of high metastatic potential subline was higher than that of low metastatic subline [(47.32 ± 3.69)% vs (35.53 ± 2.80)%, P <0.05] (61.46 ± 7.08) vs (25.32 ± 4.87) / vision, P <0.05]. Immunohistochemistry, RT-PCR and Western blot analysis showed that the expression of cortactin in the high metastatic potential cell line was higher than that in the low metastatic potential cell line (P <0.05). Conclusion The overexpression of Cortactin is closely related to the proliferation and metastasis of breast cancer.