Chu Zhang-Bo, Sun Chun-Yan, Yang Di, Chen Lei, Hu Yu
Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
J Huazhong Univ Sci Technolog Med Sci. 2013 Aug;33(4):485-490. doi: 10.1007/s11596-013-1146-3. Epub 2013 Aug 1.
This study examined the expression of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM) and its role in bone marrow angiogenesis. The peripheral blood plasma was harvested from 71 MM patients and 63 patients without hematological malignancy. The BDNF level in the blood plasma was determined by ELISA. Human bone marrow endothelial cells (HBMECs) were cultured. The mRNA and protein expression levels of the BDNF receptor TrkB in HBMECs were detected by using RT-PCR and flow cytometry, respectively. The viability of HBMECs treated with recombinant human (rh) BDNF or not was measured by using MTT assay. The migration of HBMECs in the presence of rhBDNF or not was determined by modified Boyden chamber assay. In vitro tube formation assay was used to assess the effect of rhBDNF on HBMECs differentiation. The results of ELISA revealed that the BDNF level was significantly higher in peripheral blood plasma of MM patients than in that of control patients (4.39±0.67 vs. 1.96±0.39 ng/mL, P<0.05). The BDNF receptor TrkB was expressed in HBMECs at mRNA and protein level. MTT assay manifested that rhBDNF could significantly concentration-dependently promote the HBMECs proliferation. The number of HBMECs treated with 160 ng/mL rhBDNF for 48 h was 1.57±0.10 folds higher than that in control group (P<0.05). Moreover, rhBDNF could enhance HBMECs migration in a concentration-dependent manner and the maximal migration was reached in the presence of 100 ng/mL rhBDNF. The migration indexes were 1.40±0.11, 1.64±0.16, 2.06±0.25 and 2.18±0.21 in 25, 50, 100 ng/mL rhBDNF groups and 25 ng/mL rhVEGF group, respectively. In vitro tube formation assay demonstrated that the area of the formed tubular structure was increased with the rhBDNF concentration. In control group, there was no formation of intact tubular structure and the HBMECs on the matrigel were irregularly dispersed. HBMECs treated with 100 ng/mL rhBDNF could form intact tubular structure and the area and the diameter of tubes were significantly greater than those in control group (P<0.05). There was no significant difference in the formed tubular area between 25 ng/mL VEGF group and 100 ng/mL rhBDNF group. It was concluded that BDNF plays an important role in myeloma cell-induced angiogenesis, and it may become a new target of anti-angiogenesis treatment for MM.
本研究检测了脑源性神经营养因子（BDNF）在多发性骨髓瘤（MM）中的表达及其在骨髓血管生成中的作用。收集了71例MM患者和63例无血液系统恶性肿瘤患者的外周血血浆。采用酶联免疫吸附测定法（ELISA）测定血浆中BDNF水平。培养人骨髓内皮细胞（HBMECs）。分别采用逆转录聚合酶链反应（RT-PCR）和流式细胞术检测HBMECs中BDNF受体TrkB的mRNA和蛋白表达水平。采用噻唑蓝（MTT）比色法检测重组人（rh）BDNF处理或未处理的HBMECs的活力。采用改良的Boyden小室法测定有无rhBDNF存在时HBMECs的迁移情况。采用体外成管实验评估rhBDNF对HBMECs分化的影响。ELISA结果显示，MM患者外周血血浆中BDNF水平显著高于对照组患者（4.39±0.67 vs. 1.96±0.39 ng/mL，P<0.05）。BDNF受体TrkB在HBMECs中有mRNA和蛋白水平的表达。MTT比色法显示，rhBDNF可显著浓度依赖性地促进HBMECs增殖。用160 ng/mL rhBDNF处理48 h的HBMECs数量比对照组高1.57±0.10倍（P<0.05）。此外，rhBDNF可浓度依赖性地增强HBMECs的迁移，在100 ng/mL rhBDNF存在时达到最大迁移。25、50、100 ng/mL rhBDNF组和25 ng/mL rhVEGF组的迁移指数分别为1.40±0.11、1.64±0.16、2.06±0.25和
2.18±0.21。体外成管实验表明，形成的管状结构面积随rhBDNF浓度增加而增大。对照组未形成完整的管状结构，基质胶上的HBMECs呈不规则分散。用100 ng/mL rhBDNF处理的HBMECs可形成完整的管状结构，管的面积和直径均显著大于对照组（P<0.05）。25 ng/mL VEGF组和100 ng/mL rhBDNF组形成的管状面积无显著差异。结论：BDNF在骨髓瘤细胞诱导的血管生成中起重要作用，可能成为MM抗血管生成治疗的新靶点。
