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目的:初步探讨人乳腺癌细胞MDA-MB-231特异性多肽PI的跨膜转导机制。方法:合成PI肽链,并在其N端进行FITC标记;探讨FITC-PI的跨膜转导与MHC-Ⅰ表达的关系,利用MHC-Ⅰ抗体阻断MDA-MB-231细胞和Calu-1细胞表面MHC-Ⅰ抗原,聚合酶链反应-序列特异性引物法进行基因分析;探讨FITC-PI的跨膜转导是否与小窝蛋白介导的内吞有关,抑制剂制霉菌素与MDA-MB-231细胞共培养,荧光显微镜观察以及流式细胞仪检测细胞内FITC-PI分布情况;探讨FITC-PI跨膜转导是否与2株细胞表面相同的膜蛋白有关系,膜蛋白提取试剂盒分别提取2株细胞膜蛋白,双向电泳对其进行差异性分析。结果:MHC-Ⅰ抗体阻断后,2株细胞内仍可见FITC-PI分布,基因位点分析显示2株细胞HLA-A、B位点等位基因不同;加入制霉菌素后,镜下观察及流式细胞仪检测均显示FITC-PI分布减少;双向电泳初步分析2株细胞膜蛋白图谱共有11个相同的蛋白点。结论:PI的跨膜转导机制不单一,其一,部分由小窝蛋白介导的内吞机制,其二,可能与2株细胞表面相同的膜蛋白有关系,此研究为将来继续探讨其跨膜转导机制奠定了实验基础和理论依据。
OBJECTIVE: To investigate the transmembrane transduction mechanism of PI-specific MDA-MB-231 in human breast cancer cells. Methods: The PI peptide chain was synthesized and labeled with FITC at its N-terminal. The relationship between the transmembrane transduction of FITC-PI and the expression of MHC-Ⅰ was explored. Anti-MHC-Ⅰ antibody was used to block the expression of Calu-1 Cell surface MHC-Ⅰ antigen, polymerase chain reaction-sequence-specific primer method for gene analysis; explore FITC-PI transmembrane transduction is related to caveolin-mediated endocytosis, inhibitor of nystatin and MDA- MB-231 cells were co-cultured, observed by fluorescence microscopy and flow cytometry intracellular FITC-PI distribution; explore FITC-PI transmembrane transduction with the two cell surface membrane proteins have the same relationship, membrane protein extraction kit Two cell membrane proteins were extracted and analyzed by two-dimensional electrophoresis. Results: The FITC-PI distribution was still observed in the two cells after MHC-Ⅰ antibody was blocked, and the alleles of HLA-A and B loci in the two strains were different. The addition of nystatin was observed microscopically And flow cytometry showed that the distribution of FITC-PI decreased; two-dimensional electrophoresis analysis of the two cell membrane protein profiles a total of 11 identical protein spots. Conclusion: The transmembrane transduction mechanism of PI is not unique. One of them is partly mediated by caveolin-like protein, and the other is probably related to the two membrane proteins with the same cell surface. This study will continue to explore its future Transmembrane transduction mechanism has laid the experimental foundation and theoretical basis.