Katoh-Semba R, Takeuchi I K, Semba R, Kato K
Department of Perinatology, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Japan.
J Neurochem. 1997 Jul;69(1):34-42. doi: 10.1046/j.1471-4159.1997.69010034.x.
A newly established, sensitive, two-site enzyme-immunoassay system for brain-derived neurotrophic factor (BDNF) is described. Using this system, we investigated the tissue distribution of BDNF and developmental changes in tissue levels of BDNF in rats. The minimal limit of detection of the assay was 3 pg/0.2 ml of assay mixture. BDNF was successfully solubilized from tissues in the presence of guanidine hydrochloride but not in any of the other buffers examined. In the rat brain at 1 month of age, the highest level of BDNF was detected in the hippocampus (5.41 ng/g of wet weight), followed by the hypothalamus (4.23 ng/g) and the septum (1.68 ng/g). In other regions, levels of BDNF ranged between 0.9 and 1.7 ng/g. The level of BDNF in the posterior lobes of the cerebellum from rats at 30 days of age was slightly higher than that in the anterior lobes. The concentration of BDNF increased in all regions of the brain with postnatal development. In peripheral tissues, BDNF was found at very low concentrations (0.65 ng/g in the spleen, 0.21 ng/g in the thymus, and 0.06 ng/g in the liver). The subfractionation of the hippocampal homogenate indicated that approximately 50% of BDNF was contained in the crude nuclear fraction. Immunoblots of BDNF-immunoreactive proteins extracted from the hippocampus, hypothalamus, and cerebellum contained doublet bands of protein of approximately 14 kDa, a value close to the molecular mass of recombinant human BDNF. Immunocytochemical investigations showed that, in the hippocampus, BDNF was localized in the nucleus of the granule cells in the dentate gyrus and of the cells in the pyramidal cell layer. The frequency of cells that were stained in the dentate gyrus was greater than that of cells in the pyramidal cell layer.
本文描述了一种新建立的、灵敏的、双位点酶免疫分析系统，用于检测脑源性神经营养因子（BDNF）。利用该系统，我们研究了BDNF在大鼠体内的组织分布以及BDNF组织水平的发育变化。该分析方法的最低检测限为3 pg/0.2 ml分析混合物。在盐酸胍存在的情况下，BDNF成功地从组织中溶解出来，但在所检测的其他缓冲液中均未成功溶解。在1月龄大鼠的大脑中，BDNF水平最高的部位是海马体（5.41 ng/g湿重），其次是下丘脑（4.23 ng/g）和隔区（1.68 ng/g）。在其他区域，BDNF水平在0.9至1.7 ng/g之间。30日龄大鼠小脑后叶的BDNF水平略高于前叶。随着出生后发育，大脑所有区域的BDNF浓度均升高。在周围组织中，BDNF的浓度非常低（脾脏中为0.65 ng/g，胸腺中为0.21 ng/g，肝脏中为0.06 ng/g）。海马体匀浆的亚分级分离表明，约50%的BDNF存在于粗核组分中。从海马体、下丘脑和小脑中提取的BDNF免疫反应性蛋白的免疫印迹显示，含有两条约14 kDa的蛋白条带，该值接近重组人BDNF的分子量。免疫细胞化学研究表明，在海马体中，BDNF定位于齿状回颗粒细胞的细胞核以及锥体细胞层细胞的细胞核中。齿状回中染色细胞的频率高于锥体细胞层中的细胞。
