徐红, 刘伟, 张秀珍, 侯亮, 路英进, 陈佩佩, 张璨, 冯迪, 孔力, 王秀丽
大连医科大学基础医学院机能学实验室；基础医学院组织胚胎学教研室，大连 116044；附属第二医院心血管内科，大连 116027
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。
[Development of three-dimensional breast cancer cell culture drug resistance model.] [Article in Chinese]
Xu Hong, LIU Wei, ZHANG Xiu-Zhen, HOU Liang, LU Ying-Jin, CHENG Pei-Pei, ZHANG Can, FENG Di, KONG Li, WANG Xiu-Li
Functional Laboratory； Department of Histoembryology, College of Basic Medical Sciences, Dalian Medical University, Dalian 116044, China； Department of Cardiology, The Second Hospital of Dalian Medical University, Dalian 116027, China
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 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 mRNA 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.
通讯作者：孔力,王秀丽 E-mail: firstname.lastname@example.org, email@example.com
徐红, 刘伟, 张秀珍, 侯亮, 路英进, 陈佩佩, 张璨, 冯迪, 孔力, 王秀丽. 乳腺癌细胞三维培养耐药模型的体外构建[J]. 生理学报 2016; 68 (2): 179-184.
Xu Hong, LIU Wei, ZHANG Xiu-Zhen, HOU Liang, LU Ying-Jin, CHENG Pei-Pei, ZHANG Can, FENG Di, KONG Li, WANG Xiu-Li. [Development of three-dimensional breast cancer cell culture drug resistance model.] [Article in Chinese]. Acta Physiol Sin 2016; 68 (2): 179-184 (in Chinese with English abstract).