IL-1β和TNF-α通过骨髓MSCs降低多西环素对骨髓瘤细胞株H929的细胞毒性作用  

IL-1β and TNF-α reduce cytotoxic effects of doxycycline to myeloma cell line H929 via bone marrow derived mesenchymal stem cells

作　　者：陆萍[1] 费小明[1,2] 汤郁[3] 叶炜[1] 颜玲玲[1] 朱彦[1] Lu Ping;Fei Xiaoming;Tang Yu;Ye Wei;Yan Lingling;Zhu Yan(Department of Hematology;Department of Blood Transfusion, 3Department of Rheumatism, the Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China)

机构地区：[1]江苏大学附属医院血液科,江苏镇江212001 [2]江苏大学附属医院输血科,江苏镇江212001 [3]江苏大学附属医院风湿科,江苏镇江212001

出　　处：《南京医科大学学报（自然科学版）》2018年第4期470-475,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：国家自然科学基金(81202358;81571582);江苏省卫生计生科研项目(Z201512)

摘　　要：目的:骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)在骨髓微环境中参与骨髓瘤细胞的耐药性。本研究旨在通过体外实验,研究经细胞因子预处理的骨髓MSCs在与人骨髓瘤细胞株H929共培养条件下,是否影响多西环素(doxycycline,DOX)对骨髓瘤细胞株的细胞毒性作用,并初步探讨其可能的作用机制。方法:经白细胞介素(interleudin,IL)-1β或肿瘤坏死因子(tumor necrosis factor,TNF)-ɑ预处理的人骨髓MSCs,与H929细胞共培养,并以DOX处理细胞,分别采用CCK8法和流式细胞术(FCM),检测DOX对H929细胞增殖和凋亡的影响;采用FCM和实时荧光定量PCR(RT-q PCR)法检测IL-1β或TNF-α处理后,骨髓MSCs中血管细胞黏附分子(VCAM-1)表达;Western blot法检测在DOX存在时,不同处理的骨髓MSCs作为共培养饲养层细胞的条件下,H929细胞p-Erk1/2的变化。结果:DOX可抑制骨髓瘤细胞株H929的增殖并诱导其凋亡。人骨髓MSCs与H929共培养可以减少DOX对H929的细胞毒性作用,而经IL-1β或TNF-α预处理的人骨髓MSCs再与H929共培养,则可以进一步降低DOX的细胞毒性作用。人骨髓MSCs经IL-1β或TNF-α处理后,其VCAM-1 m RNA转录水平和蛋白表达水平均升高。经预处理的骨髓MSCs与H929共培养后,可以降低DOX对骨髓瘤细胞株p-Erk1/2的下调作用。结论:DOX在体外对骨髓瘤细胞株表现出剂量和时间依赖性的细胞毒性作用;而IL-1β和TNF-α可通过骨髓MSCs来间接拮抗DOX对H929的细胞毒性作用;IL-1β和TNF-α的这一作用机制可能与上调骨髓MSCs的VCAM-1表达与上调p-Erk1/2有关。Objective：Bone marrow derived mesenchymal stem cells（MSCs）in bone marrow microenvironment was found to be involved in the chemoresistance of myeloma cells. In this study,we investigate the role of IL-1β or TNF-α-treated bone marrow MSCs in chemosensitivity of myeloma cell line H929 to doxycycline（DOX）in vitro,and find some possible mechanisms. Methods：CCK8 assay was employed to measure the proliferative rate of H929 cells in the presence of DOX at different concentration,either alone or co-culture with bone marrow MSCs pretreated with IL-1β or TNF-α or not. The apoptotic ratio of H929 cells treated with DOX in the presence of IL-1β or TNF-α-treated bone marrow MSCs or not was determine by flow cytometry（FCM）. FCM was also used with real time fluorescence quantitative polymerase chain reaction（RT-qPCR）to measure vascular cell adhesion molecule type one（VCAM-1）on MSCs. The p-Erk1/2 expression level in H929 was measured by Western blot. Results：DOX inhibited the proliferation and induced apoptosis of H929 in a time and dose-dependent manner. IL-1β or TNF-α-pretreated bone marrow MSCs reduced the cytotoxic effects of DOX in H929 cells. Expression level of p-Erk1/2 was down-regulated in H929 cells in the presence of DOX treatment,and this down-regulating effect of DOX was most pronounced when H929 cells were co-cultured with IL-1β or TNF-α-pretreated bone marrow MSCs. In addition,we found that VCAM-1 expression of bone marrow MSCs was up-regulated by IL-1β or TNF-α treatment. Conclusion：DOX was shown to have cytotoxicity to myeloma cells line H929 in a time and dose-dependent manner in vitro. IL-1β or TNF-α could abrogate the cytotoxic effects of DOX in H929 cells indirectly via bone marrow MSCs. Erk pathway in H929 cells and VCAM-1 expression on MSCs may be involved in this myeloma-protective effects by IL-1β or TNF-α.

关 键 词：多发性骨髓瘤 多西环素 骨髓间充质干细胞 IL-1Β TNF-Α P-ERK1/2 

分 类 号：R733.3[医药卫生—肿瘤]