多发性骨髓瘤细胞脑源性神经营养因子对血管新生作用的研究  

Effect of brain-derived neurotrophic factor in human myeloma cells on angiogenesis

作　　者：孙春艳[1] 胡豫[1] 吴涛[1] 王雅丹[1] 何文娟[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》2005年第10期602-606,共5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：目的研究多发性骨髓瘤(MM)细胞脑源性神经营养因子(BDNF)的表达水平及其与MM细胞诱导的血管新生的关系。方法采用RT-PCR法、Western blot法及ELISA法检测MM细胞系KM3、RPMI8226细胞BDNF的表达及分泌。采用MTT法观察MM细胞培养上清液对脐静脉内皮细胞(HUVEC)增殖的作用;用改良的Boyden小室法观察MM细胞培养上清液对HUVEC迁移的影响;采用体外小管形成实验,观察MM细胞培养上清液对HUVEC分化的影响。结果KM3、RPMI8226细胞不仅表达BDNF mRNA,也表达和分泌BDNF蛋白,BDNF的基础分泌水平在其生物学作用范围内。KM3、RPMI8226细胞培养上清液均可明显促进HUVEC增殖,含50.0%KM3细胞培养上清液组和完全KM3细胞培养上清液组HUVEC数分别为对照组的(1.85±0.23)倍和(2.16±0.29)倍(P<0.05),抗人类BDNF中和抗体可部分抑制其促增殖活性;含50.0%KM3细胞培养上清液组HUVEC迁移指数为1.85±0.23,完全KM3细胞培养上清液组HUVEC迁移指数为2.16±0.29,与对照组比较,差异有统计学意义(P值均<0.05),并可明显促进基质胶中网状毛细血管形成(P<0.01),抗BDNF中和抗体可明显抑制其作用。结论MM细胞表达和分泌BDNF,BDNF可能参与MM细胞诱导的血管新生。Objective To investigate the expression of brain-derived neurotrophic factor （BDNF） in multiple myeloma （MM） cells and the correlation between BDNF and MM angiogenesis. Methods The expressions of BDNF mRNA transcripts and protein in MM cell lines （ RPMI 8226, KM3） were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay. Proliferation of human umbilical vein endothelial cells （HUVEC） mixed with MM cuhure medium at different concentrations was examined by MTT assay. The effects of MM culture medium on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively. Results BDNF was expressed in and secreted by MM cell lines RPMI 8226 and KM3. BDNF concentrations in culture supernatants were within the range of its biological activity. MM culture medium induced a concentration-dependent proliferation of HUVEC. The number of HUVEC at a concentration of 50% KM3 culture medium and at full KM3 culture medium were （ 1.85 ± 0.23 ） -fold and （ 2.16 ± 0.29） -fold increase, respectively （P 〈0.05） , compared with that of control. The proliferative activity of HUVEC was reduced on the addition of BDNF antibody to the culture medium. MM culture medium also stimulated the migration and differentiation of HUVEC in vitro, the chemotactic index of HUVEC at a concentration of 50% KM3 culture medium and at full KM3 culture medium were 1.85±0.23 and 2.16 ± 0.29, respectively（ P 〈 0.05）. Full KM3 culture medium also stimulated capillary-like tube formation in HUVEC （ P 〈0.01 ） , and addition of anti-human BDNF antibody neutralized these effects significantly. Conclusion MM cell lines expressed and secreted biologically active BDNF, which may be involved, at least in part, in MM angiogenesis.

关 键 词：多发性骨髓瘤 脑源性神经营养因子 血管新生 多发性骨髓瘤细胞 细胞培养上清液 BDNF蛋白 HUVEC ELISA法检测 BOYDEN小室 RT-PCR法 

分 类 号：R733.3[医药卫生—肿瘤]