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本研究旨在体外构建乳腺癌三维(3D)培养模型,部分模拟肿瘤细胞在体微环境,以期建立理想的乳腺癌耐药研究模型。将乳腺癌MCF-7细胞与胶原支架材料复合,体外构建乳腺癌3D培养模型。用Carmine和HE染色法观察细胞形态,用CCK-8法检测细胞增殖情况,用活-死细胞染色试剂盒观察细胞活性,检测3D培养和二维(2D)培养条件下细胞对阿霉素、卡铂、5-氟尿嘧啶的药物敏感性,实时定量RT-PCR检测耐药相关基因P-糖蛋白(P-glycoprotein,P-gp)和多药耐药相关蛋白2(mrp2)m RNA表达水平,用Western blot检测P-gp蛋白的表达。结果显示,3D培养组内MCF-7细胞呈聚团生长,细胞增殖及活性良好。和2D培养组相比,3D培养组细胞对化疗药物的敏感性显著降低,P-gp和mrp2 m RNA水平显著升高,P-gp蛋白水平升高至5.3倍。以上结果提示,本研究成功构建了具有耐药表型及功能的乳腺癌3D培养模型。该模型对深入阐明耐药发生相关机制等研究具有重要意义。
The aim of this study was to construct a three-dimensional (3D) culture model of breast cancer in vitro and partially to simulate the in vivo microenvironment of tumor cells in order to establish an ideal model for the study of drug resistance in breast cancer. Breast cancer MCF-7 cells and collagen scaffold materials, in vitro construction of breast cancer 3D culture model. Cell morphology was observed by Carmine and HE staining. Cell proliferation was detected by CCK-8 assay. Cell viability was detected by live-dead cell staining kit. The effects of adriamycin, Carboplatin and 5-fluorouracil. The mRNA expression levels of P-glycoprotein (P-gp) and mrp2 (mrp2) were detected by real-time quantitative RT- Western blot was used to detect the expression of P-gp protein. The results showed that the MCF-7 cells in the 3D culture group grew agglomerate, with good cell proliferation and activity. Compared with the 2D culture group, the sensitivity of the cells in 3D culture group to chemotherapeutic drugs was significantly reduced, the P-gp and mrp2 m RNA levels were significantly increased, and the P-gp protein level was increased to 5.3 times. The above results suggest that this study successfully constructed a 3D model of breast cancer with resistant phenotype and function. This model is of great importance to further elucidate the mechanisms of drug resistance.