脑源性神经营养因子激活其受体TrkB在多发性骨髓瘤发病机制中的作用

Study of brain-derived neurotrophic factor activating TrkB signaling cascades in the pathogenesis of multiple myeloma

目的研究异常表达的脑源性神经营养因子（BDNF）在多发性骨髓瘤（MM）发生、发展中的作用及其信号通路。方法采用锥虫蓝拒染法检测BDNF对人MM细胞系RPMI8226、U266和KM3细胞存活的促进作用，MTT比色法观察BDNF对苯丙氨酸氮芥和长春新碱细胞毒作用的影响，Western blot检测BDNF对MM细胞TrkB磷酸化的影响。通过动物模型观察BDNF对肿瘤生长和实验动物存活时间的影响。结果BDNF以浓度依赖性方式促进MM细胞的存活，50μg／LBDNF作用后存活细胞数较对照组明显增加。BDNF可明显降低苯丙氨酸氮芥和长春新碱的细胞毒性，50μg／LBDNF作用后，苯丙氨酸氮芥和长春新碱的EC50值分别为无BDNF作用时的2倍和3倍。体内实验表明BDNF可明显促进异种移植的MM裸鼠模型中肿瘤的生长，经BDNF处理和未经BDNF处理组肿块的平均体积分别为3240．9mm^3和1032．7mm^3（P〈0．05），生存时间分别为13d和21d（P〈0.05）。MM细胞中异常表达的TrkB是BDNF的功能性受体，外源性BDNF作用后，MM细胞TrkB磷酸化水平明显增加，并且Trk抑制剂K252a可明显抑制BDNF诱导的MM细胞迁移。结论外源性BDNF可磷酸化激活MM细胞表面TrkB受体，促进骨髓瘤细胞的增殖、存活、迁移并参与骨髓瘤细胞的化疗耐药，在MM的病理生理进程中起重要作用。

Objective To explore the significance of abnormal expression of brain-derived neurotrophic factor（ BDNF）/TrkB in the development and evolution of multiple myeloma（MM） and the involved signaling pathways. Methods The effect of BDNF on the cell viability of human myeloma cell line （HMCL） （RPMI8226, U266, KM3） was determined by trypan blue dye-exclusion. MTr assay was used to evaluate the cytotoxicity of tested chemotherapeutic agents. The effect of BDNF on the pbospborylation of TrkB was determined by Western blot. A human myeloma xenograft animal model was used to evaluate the effects of BDNF on tumor growth and survival time. Results BDNF at 50 μg/L triggered significant increase in cell viability of HMCL. BDNF protected KM3 cells from melphalan and vincristine. The viability of KM3 cells exposed to varying concentrations of melphalan with and without 50 μg/L BDNF showed that BDNF induced almost a 2-fold and a 3-fold increase in melphalan and vincristine toxicity respectively. BDNF treatment increased MM cell growth in xenografted MM model （3240.9 mm^3 vs 1032.7 mm^3 ） （ P 〈0.05）. Intratumoral injection of BDNF also significantly reduced survival time （ 13 d vs 21 d） （P 〈 0.05 ）. The phosphorylated TrkB level was increased significantly after treated by exogenous BDNF. BDNF-triggered migration in RPMI8226 cells was completely abrogated by a Trk tyrosine kinase inhibitor K252a. Conclusion BDNF can activate TrkB signaling cascades resulting in MM cells growth, migration, and chemoprotection and appears to have a major contribution to the patbogenesis of MM.