人骨髓间充质细胞在低氧培养条件下脑源性神经营养因子的表达

Expression of brain-derived neurotrophic factor in human bone marrow stromal cells cultured in hypoxia

目的:观察低氧培养条件下人骨髓间充质细胞中脑源性神经营养因子的表达情况,为其用于脑缺血治疗提供依据。方法:实验于2005-09/12在暨南大学医学院血液病研究所完成。生长至次融合状态的第5代人骨髓间充质细胞在低氧混合气(体积分数0.90氮气、体积分数0.05二氧化碳和体积分数0.05氧气)条件下培养,应用半定量反转录聚合酶链反应和酶联免疫技术检测其脑源性神经营养因子基因表达。结果:①骨髓间充质细胞常氧压培养时表达脑源性神经营养因子mRNA和蛋白水平低:半定量反转录聚合酶链反应检测脑源性神经营养因子mRNA与β-肌动蛋白mRNA信号百分比为(6.35±2.39)%,酶联免疫法检测脑源性神经营养因子蛋白为(137.6±12.3)ng/L。②低氧培养显著增加骨髓间充质细胞表达脑源性神经营养因子mRNA和蛋白水平:低氧培养2h时脑源性神经营养因子mRNA与β-肌动蛋白mRNA信号百分比明显高于常氧压培养时表达水平,8h时达到高峰水平[低氧培养2h:(13.94±3.07)%,F=14.66,P<0.01;8h:(27.49±7.18)%]。低氧培养4h时脑源性神经营养因子蛋白显著增高,明显高于常氧培养组,16h时达到高峰水平[低氧培养4h:(165.6±13.9)ng/L,F=11.70,P<0.01;16h:(237.4±15.1)ng/L]。结论:合适低氧条件可增强骨髓间充质细胞表达脑源性神经营养因子,提示其在脑缺血组织等低氧环境下可能产生脑源性神经营养因子样生物学作用,可用于治疗脑缺血等疾病。

AIM： To observe the expression of brain-derived neurotrophic factor （BDNF） in bone marrow stromal cells cultured in hypoxia, so as to provide evidences for its application in treatment of brain isehemia. METHODS： The experiment was carried out in the Institute of Hematology, Medical College of Jinan University from September to December 2005. Subconfluent cells of the fifth passage were cultured in hypoxia mixture gas （composed of nitrogen gas with volume fraction of 0.90, carbon dioxide with volume fraction of 0.05 and oxygen with volume fraction of 0.05）. BDNF mRNA was detected by semi-quantitative RT-PCR, and BDNF protein by ELISA. RESULTS： ①B0ne marrow stromal cells expressed low levels of BDNF mRNA and protein in normal culture condition： Semi-quantitative RT-PCR showed that the ratio of BDNF to β-actin was （6.35±2.39）%, and ELISA showed the BDNF protein was （137.6±12.3） ng/L. ②Hypexia enhanced the expression of BDNF mRNA and BDNF protein： After 2 hours of cell culture in hypoxia condition, the ratio of BDNF to β-actin was significantly higher than that in the normal culture group, and it reached the peak level at the 8th hour [the 2nd hour： （13.94±3.07）%, F=14.66, P〈0.01; the 8th hour： （27.49±7.18）%]. At the 4th hour under hypoxia, the BDNF protein significantly increased and was higher than that in the normal culture group; at the 16th hour in hypoxia, BDNF protein reached, the peak [the 4th hour： （165.6±13.9） ng/L, F=ll.70, P〈0.01; the,16th hour： （237.4±15.1） ng/L]. CONCLUSION： Appropriate hypoxia can enhance the expression of BDNF mRNA and BDNF in bond marrow stromal cells, which may display BDNF-like biological effect in hypoxia condition like cegebral isehemic tissues, and can be used in therapy of cerebral isehemia.