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本实验采用凝胶过滤层析法和高效液相色谱技术,分离纯化备用根大鼠部分去传入侧的脊髓后角组织中的神经营养活性物质.1.生物活性检测方法的建立:切除大鼠一侧背根和背根节(DRG),仅保留L-4背根为备用根.术后动物存活5天.分别切取L1-L6节段手术侧和非手术侧脊髓后角组织,制备含手术侧和非手术侧提取液的培养液.用此液分别培养鸡胚DRG,48小时后手术侧组DRG神经突起密度明显大于对照组和非手术侧组,提示去传入纤维支配的脊髓后角组织存在着促神经突起生长的神经营养活性物质.又采用改良的悬滴培养法,发现手术侧脊髓后角组织能支持鸡胚DRG神经元的存活,而且具有促进神经突起生长的作用.2.备用根大鼠脊髓后角组织神经营养活性物质的分离与纯化,部分背根切除术后5天处死动物,切取手术侧脊髓L1-L6节段后角组织,匀浆、离心后,取上清液经Superdex prep grade G-75凝胶过滤层析,呈现Ⅰ、Ⅱ二个洗脱峰,将各洗脱峰浪加入培养基,Ⅰ峰洗脱液表现有神经营养作用.将Ⅰ洗脱峰进行SDS-聚丙烯酰胺凝胶电泳分析,银染后呈现多条明显的蛋白区带,去传入脊髓后角组织呈现神经营养活性的物质是在40-78KD之间.将Ⅰ峰洗脱液浓缩,进行高效液相色谱(HPLC)分析,得到4个不同的洗脱峰(a、b、c、d峰).将各洗脱峰液配制成不同浓度的
In this experiment, gel filtration chromatography and high performance liquid chromatography were used to separate and purify the neurotrophic substances in the descending horn of spinal cord from the part of the spare root to the afferent side.1.Development of biological activity detection method: Rats were dorsal root and dorsal root ganglion (DRG), and only L-4 dorsal root was reserved as spare root.The animals survived for 5 days.After surgical resection of L1-L6 segment, The culture medium containing both surgical and non-surgical extracts was used to culture chick embryo DRG. After 48 hours, the density of DRG neurites in the surgical side group was significantly larger than that in the control and non-surgical side groups, In the posterior horn tissue, neurotrophic substances that promote the growth of neurites are present, and by the modified hanging drop culture method, it is found that the posterior horn tissue of the spinal cord on the operative side can support the survival of DRG neurons in chicken embryos and promote the growth of neurites. The separation and purification of neurotrophic substances in the spinal dorsal horn tissue of spare root rats were performed. The animals were sacrificed 5 days after partial dorsal rhizotomy and the posterior horn of L1-L6 segments of the spinal cord were excised, homogenized and centrifuged The supernatant was subjected to Superdex prep gra De G-75 gel filtration chromatography, showing Ⅰ, Ⅱ two eluting peaks, the eluting peak wave was added to the medium, Ⅰ peak eluent showed neurotrophic effect of Ⅰ eluting peak SDS- poly Acrylamide gel electrophoresis analysis, silver staining showed a number of obvious protein bands, to pass into the spinal cord posterior horn tissue showing neurotrophic activity substance is between 40-78KD.I peak eluate concentration, efficient Four different elution peaks (a, b, c, d peaks) were obtained by liquid chromatography (HPLC) analysis.Each elution peak was prepared in different concentrations