College of Oral Medicine, Taipei Medical University, Taipei, Taiwan.
Transfusion. 2012 Aug;52(8):1721-8. doi: 10.1111/j.1537-2995.2011.03494.x. Epub 2011 Dec 29.
Human blood platelets (PLTs) contain brain-derived neurotrophic factor (BDNF), a neurotrophin that binds to neurotrophic tropomyosin-related kinase B (TrkB) receptor on central nervous system cells. This binding promotes neural synaptic plasticity and memory and prevents neuronal degeneration. Alterations in BDNF homeostasis are associated with aging and are found in several neurodegenerative conditions such as Alzheimer's, Huntington's, and Parkinson's diseases and multiple sclerosis. We have developed PLT viral inactivation and chromatographic fractionation processes and decided here to identify fractions enriched in BDNF.
PLT concentrates (PCs) were treated by solvent/detergent (S/D), extracted by oil, and subjected to fractionation (C18, sulfopropyl [SP]-Sepharose, diethylaminoethyl [DEAE]-Sepharose, or activated charcoal). BDNF and pro-BDNF were evaluated by enzyme-linked immunosorbent assay, and Western blot. TrkB was studied by Western blot. Tri-n-butyl phosphate (TnBP) was quantified by high-performance liquid chromatography, and Triton X-45 by gas chromatography.
The mean BDNF content of 2.9 ± 0.7 ng/mL in PC was noted to increase to 56.2 ± 2.4 ng/mL after S/D treatment and remained stable during oil extraction. Approximately 70% of the BDNF content was recovered after C18 chromatography. BDNF did not bind to DEAE-Sepharose and was almost completely adsorbed by charcoal. Chromatography on SP-Sepharose yielded a highly enriched 13-kDa mature BDNF fraction that was more than 170-fold purified, with a mean of 137 ± 29.4 ng/mL and 82% chromatographic recovery, devoid of detectable TnBP and Triton X-45. Pro-BDNF and TrkB proteins were not detected in the PLT extracts.
We obtained a S/D-treated, highly enriched mature PLT-derived BDNF fraction that could help unveil the pharmacokinetics, pharmacodynamic, and potential therapeutic applications of the BDNF neurotrophin.
人类血小板（PLT）中含有脑源性神经营养因子（BDNF），这是一种神经营养素，可与中枢神经系统细胞上的神经营养素原肌球蛋白相关激酶 B（TrkB）受体结合。这种结合促进神经突触可塑性和记忆，并防止神经元变性。BDNF 动态平衡的改变与衰老有关，并且在几种神经退行性疾病中都有发现，如阿尔茨海默病、亨廷顿病、帕金森病和多发性硬化症。我们已经开发出血小板病毒灭活和色谱分离工艺，在这里决定鉴定富含 BDNF 的部分。
PLT 浓缩物（PC）经溶剂/去污剂（S/D）处理，用油提取，并进行分级（C18、磺丙基[SP]-琼脂糖、二乙基氨基乙基[DEAE]-琼脂糖或活性炭）。BDNF 和 pro-BDNF 通过酶联免疫吸附试验和 Western blot 进行评估，TrkB 通过 Western blot 进行研究。通过高效液相色谱法测定三丁基磷酸酯（TnBP）的含量，通过气相色谱法测定 Triton X-45 的含量。
PC 中平均 2.9±0.7ng/mL 的 BDNF 含量在 S/D 处理后增加到 56.2±2.4ng/mL，在油提取过程中保持稳定。C18 色谱后约 70%的 BDNF 含量得到回收。BDNF 不与 DEAE-琼脂糖结合，几乎完全被活性炭吸附。SP-琼脂糖色谱得到高度富集的 13kDa 成熟 BDNF 级分，纯化度超过 170 倍，平均含量为 137±29.4ng/mL，回收率为 82%，无检测到 TnBP 和 Triton X-45。PLT 提取物中未检测到 pro-BDNF 和 TrkB 蛋白。
我们获得了一种 S/D 处理的、高度富集的成熟血小板衍生 BDNF 级分，这可能有助于揭示 BDNF 神经营养素的药代动力学、药效学和潜在治疗应用。
