【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。更多还原

【Abstract】 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 coculture 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-q PCR)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.更多还原