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目的探讨皮质肌动蛋白(Cort)对高表达Cort的乳腺癌细胞系(MDA-MB-231细胞)细胞内吞作用的影响。方法在MDA-MB-231细胞中使用Cort siRNA干预技术和“生物搬运法”系统将抗Cort免疫抗体(Cort多克隆抗体001或单克隆Cort抗体4F11)引入MDA-MB-231细胞,进行体内干预Cort功能。根据捕获ELISA法分析转铁蛋白摄入,免疫印迹分析细胞中Cort敲除的程度和免疫荧光直接检测评价Cort在转铁蛋白吸收中的作用。结果免疫印迹分析在Cort siRNA处理过的细胞质中未测出Cort蛋白;用荧光标记评价转铁蛋白密度水平,在Cort siRNA处理的细胞中要低于含相对高水平Cort的对照组细胞;在接受Cort siRNA处理后仅50%的MDA-MB-231细胞中有转铁蛋白摄入;在MDA-MB-231细胞中引入001或4F11抗体,其细胞的内吞作用减弱,导致明显的转铁蛋白吸收减少30%~60%。结论干扰Cort的功能或直接使用抗Cort抗体进行干预都能抑制细胞内吞作用。Cort是细胞内吞作用中必不可少的成分,具有重要作用。
Objective To investigate the effect of cortin on endocytosis in Cort-overexpressing breast cancer cell lines (MDA-MB-231 cells). Methods Anti-Cort immune antibody (Cort polyclonal antibody 001 or monoclonal Cort antibody 4F11) was introduced into MDA-MB-231 cells using Cort siRNA interference technology and the “Biotransport” system in MDA-MB-231 cells. In vivo intervention Cort function. Transferrin uptake was analyzed by capture ELISA and the extent of Cort knockdown in the cells was analyzed by immunoblotting and the direct immunofluorescence assay was used to assess Cort’s role in transferrin uptake. Results Western blot analysis No Cort protein was detected in the Cort siRNA-treated cytoplasm; transferrin densities were assessed by fluorescent labeling, which was lower in Cort siRNA-treated cells than in control cells containing relatively high levels of Cort; There was transferrin uptake in only 50% of MDA-MB-231 cells after Cort siRNA treatment; the incorporation of 001 or 4F11 antibodies into MDA-MB-231 cells resulted in diminished endocytosis of cells, leading to significant transferrin Absorption reduced by 30% to 60%. Conclusions Interfere with the function of Cort or directly with anti-Cort antibody can inhibit the endocytosis. Cort is an essential component of endocytosis and plays an important role.