乳腺癌细胞三维培养耐药模型的体外构建  

Development of three-dimensional breast cancer cell culture drug resistance model

作　　者：徐红[1] 刘伟[2] 张秀珍[2] 侯亮[3] 路英进 陈佩佩[2] 张璨[2] 冯迪[2] 孔力[2] 王秀丽[2] 

机构地区：[1]大连医科大学基础医学院机能学实验室,大连116044 [2]大连医科大学基础医学院组织胚胎学教研室,大连116044 [3]大连医科大学附属第二医院心血管内科,大连116027

出　　处：《生理学报》2016年第2期179-184,共6页Acta Physiologica Sinica

基　　金：supported by the National Natural Science Foundation of China(No.31470952);Natural Science Foundation of Liaoning Province;China(No.2014023003)

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)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 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 m RNA 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 3D culture formed a number of cell aggregates, and most of them displayed good cell viability. The IC50 values of doxorubicin, carboplatin, 5-fluorouracil were all increased significantly in 3D culture compared with those in 2D culture. Moreover, compared with MCF-7 cells in 2D culture, the cells in 3D culture showed increased m RNA levels of P-gp and mrp2, as well as up-regulated protein expression of P-gp. These results suggest that in vitro collagen-embedded culture system of human breast cancer cells represents an improved pathologically relevant 3D microenvironment for breast cancer cells, providing a robust tool to explore the mechanism of drug resistance of cancer cells.

关 键 词：MCF-7 3D培养 胶原支架材料 耐药 

分 类 号：R737.9[医药卫生—肿瘤]