乳腺癌细胞三维培养耐药模型的体外构建.pdfVIP

生理学报 Acta Physiologica Sinica, April 25, 2016, 68(2): 179–184 DOI: 10.13294/j.aps.2016.0017 179 乳腺癌细胞三维培养耐药模型的体外构建 徐 红1，刘 伟2，张秀珍2，侯 亮3，路英进3，陈佩佩2，张 璨2，冯 迪2，孔 力2,*，王秀丽2,* 大连医科大学1基础医学院机能学实验室；2基础医学院组织胚胎学教研室，大连 116044；3附属第二医院心血管内科，大连 116027 摘 要：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) mRNA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 mRNA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。 关键词：MCF-7；3D培养；胶原支架材料；耐药 中图分类号：R3 Development of three-dimensional breast cancer cell culture drug resistance model XU Hong1, LIU Wei2, ZHANG Xiu-Zhen2, HOU Liang3, LU Ying-Jin3, CHEN Pei-Pei2, ZHANG Can2, FENG Di2, KONG Li2,*, WANG Xiu-Li2,* 1Functional Laboratory; 2Department of Histoembryology, College of Basic Medical Sciences; Dalian Medical University, Dalian 116044, China; 3Department of Cardiology, The Second Hospital of Dalian Medical University, Dalian 116027, China Abstract: The aim of the present study was to develop three-dimensional (3D) culture model, a more pathologically relevant model, of human breast cancer for drug resistance study. MCF-7 cells were embedded within collagen gel to establish 3D culture model. Cellular morphology was observed using Carmine and HE staining. Cell proliferation was evaluated by CCK-8 assay, and cell activity was detected by Live/Dead staining kit. Drug sensitivities of the 3D culture to doxorubicin, carboplatin, 5-fluorouracil were assayed and compared with those of monolayer (2D) culture. In addition, the levels of drug resistance-related genes P-glycoprotein (P-gp), mrp2 mRNA expressions were detected by real time RT-PCR. Expression level of P-gp protein was detected by Western blot. The results showed that MCF-7 cells in