多发性骨髓瘤细胞激活内皮细胞脑源性神经营养因子自分泌环  

Human multiple myeloma cell line RPMI8226 activates brain derived neurotrophic factor autocrine loop of co-cultured endothelial cells

作　　者：王雅丹[1] 胡豫[1] 孙春艳[1] 何文娟[1] 张小平[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院血液病研究所,武汉430022

出　　处：《中华血液学杂志》2007年第4期235-238,共4页Chinese Journal of Hematology

基　　金：湖北省青年杰出人才基金资助项目（2003ABB017）.

摘　　要：目的在体外共培养体系中研究人多发性骨髓瘤（MM）细胞对正常内皮细胞的作用。方法建立人MM细胞株RPMI8226与正常人脐静脉内皮细胞（HUVEC）的体外共培养体系；以同期单独培养的HUVEC为对照，对与RPMI8226细胞共培养的HUVEC进行脑源性神经营养因子（BDNF）及其特异性受体TrkB基因的半定量RT—PCR分析和蛋白的Western blot分析，采用ELISA方法检测共培养体系培养上清中BDNF的含量；在转换共培养实验的基础上应用Transwell小室迁移实验、网状结构形成实验评价与RPMI8226细胞共培养激活的HUVEC对正常HUVEC血管新生能力的影响。结果与同期单独培养的HUVEC相比，经共培养后的HUVEC不仅上清中BDNF的含量增加[分别为（12．4±5．1）ng／ml和（31．6±7．2）ng／ml，P〈0．05]，RT-PCR结果显示HUVEC常规表达BDNF，与RPMI8226细胞共培养后HUVEC BDNF表达量约为前者的1．7倍（P〈0．05）；单独培养的HUVEC几乎不表达TrkB，共培养的RPMI8226细胞明显上调HUVEC TrkB的表达（为前者的4．4倍，P〈0．05），Western blot结果与上述结果相符；经RPMI8226细胞活化的内皮细胞可明显促进HUVEC迁移和网状结构形成，与未活化的内皮细胞相比迁移指数和网状结构数量分别增加了99％和72％，抗BDNF抗体可部分抑制其活性。结论MM细胞通过可溶性的细胞因子介导，激活内皮细胞的BDNF／TrkB自分泌环，继而实现内皮细胞的自促进血管新生效应。Objective To study the influence of multiple myeloma cells on normal endothelial cells in co-culture system. Methods Human multiple myeloma cell line RPMI8226 was co-cultured with human umbilical vein endothelial cells （HUVECs）. HUVECs cultured alone were used as control. The expression of brain derived neurotrophic factor （BDNF） and its specific acceptor TrkB mRNA and protein in HUVECs were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay（ELISA）. After transferring the co-culture, the effects RPMI8226 on HUVECs angiogenesis were studied by modified transwell migration assay and net-like formation assay. Results The median BDNF concentration in culture supernatant was increased in co-cultured HUVECs compared with that in HUVECs cultured alone [ （ 31.6 ± 7.2） ng/ml vs （ 12.4 ± 5.1 ） ng/ml, P 〈 0.05 ]. The expression of BDNF transcript demonstrated by RT-PCR did the same in the two culture systems （ 1.7 fold increase, P 〈 0.05 ）. TrkB mRNA was hardly detected in culture of HUVECs alone but was increased in co-cultured HUVECs （4.4- fold increase, P 〈 0.05 ）. The BDNF and TrkB protein expressions determined by Western blot were similar to that of their mRNAs. On the other hand, the RPMI8226 activated HUVECs showed enhanced migration and net-like formation, being increased by 99% and 72%, respectively. Addition of anti-human BDNF antibody to the culture medium partly reduced these effects. Condusion Multiple myeloma cells activated BDNF/TrkB autocrine loops in co-cultured endothelial cells and resulted in endothelial self-activating angiogenesis.

关 键 词：多发性骨髓瘤 细胞培养 脑源性神经营养因子 血管新生 

分 类 号：R686[医药卫生—骨科学]