肿瘤相关巨噬细胞诱导乳腺癌耐药的实验研究

Investigation on the determination of tumor-associated macrophages inducing the drug resistance of human breast cancer

目的探索肿瘤相关巨噬细胞(TAM)在乳腺肿瘤细胞耐药中的作用及其耐药机制。方法通过佛波酯(PMA)、白细胞介素4(IL-4)、白细胞介素13(IL-13)刺激人单核细胞白血病细胞系THP-1,建立TAM模型在体外与乳腺癌细胞共培养,运用流式细胞术(FCM)检测TAM表面分子CD14、CD204;使用噻唑蓝(MTT)比色法检测紫杉醇对乳腺癌细胞系T47D、BT-549的有效杀伤浓度和时间,以及乳腺癌细胞单独培养、乳腺癌细胞与TAM/TAM上清共培养下乳腺癌细胞的杀伤;运用Western blot检测肿瘤细胞的磷酸化信号转导与转录激活因子3(p-STAT3)、磷酸化c-jun氨基末端激酶(p-JNK)信号通路变化。结果 THP-1细胞经PMA、IL-4、IL-13诱导分化为TAM,其细胞表面表达CD14、CD204(表达率分别为31.52%±9.39%、48.21%±8.76%)。TAM/TAM上清与肿瘤细胞共培养均可显著削弱紫杉醇对肿瘤细胞的杀伤(T47D相对存活率由43.52%±9.40%提高至92.68%±2.32%/92.20%±2.10%,BT-549相对存活率由63.08%±2.71%提高至77.96%±2.64%/79.55%±2.35%,P<0.05),明显下调其凋亡信号p-JNK(P<0.05),同时显著上调p-STAT3(P<0.05)。结论成功建立TAM模型,TAM介导乳腺肿瘤细胞耐药,其机制可能与TAM分泌的细胞因子影响肿瘤细胞STAT3、JNK信号分子磷酸化有关。

Objective To investigate the role and drug resistance mechanism of tumor-associated macrophages( TAM) in human breast cancer cells. Methods Human monocytic cell line THP-1 was stimulated by phorbol myristate acetate( PMA),interleukin 4( IL-4) and interleukin 13( IL-13) to build a model of TAM which was cocultured with breast cancer cells in vitro. Flow cytometry( FCM) was used to determine CD14 and CD204. The effective concentration and time of paclitaxel for inhibiting the proliferation of breast cancer cell line T47 D and BT-549,and the inhibitory rates of breast cancer cell cultured alone and cocultured with TAM /TAM conditioned medium were detected by3-( 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide( MTT) colorimetric signal. The signal pathway change of phosphorylated signal transducers and activators of transduction 3( p-STAT3) and phosphorylated c-Jun N-terminal kinase( p-JNK) were analyzed by Western Blot. Results THP-1 differentiated to TAM by PMA,IL-4 and IL-13 and showed the expressions of CD14( 31. 52% ± 9. 39%) and CD204( 48. 21% ± 8. 76%). Breast cancer cell lines which had been cocultured with TAM /TAM conditioned medium showed a significant increase in the number of survival cells compared to breast cancer cell lines cultured alone( T47 D cultured alone: 43. 52% ± 9. 40%,T47 D cocultured:92. 68% ± 2. 32% /92. 20% ± 2. 10%,BT-549 cultured alone: 63. 08% ± 2. 71%,BT-549 cocultured: 77. 96% ±2. 64% /79. 55% ± 2. 35%,P < 0. 05). The apoptosis signal p-JNK was significantly down-regulated( P < 0. 05),and the p-STAT3 was up-regulated( P < 0. 05). Conclusions The model for TAM is established successfully. TAM protects breast cancer cells from the effect of paclitaxel through the secretion of cytokines which may caused by the activation of STAT3 and down regulation of p-JNK in tumor cells.