雪旺细胞分泌的>10kD蛋白分子支持脊髓前角神经元生存的初步报告

>10kD Proteins Secreted from Schwann Cell Supports the Survival of Spinal Anterior Horn Neuron

目的：探讨成年大鼠坐骨神经瓦勒变性期雪旺细胞（ＳＣ）促进周围神经再生的物质基础。方法：将２１５ｍｌＳＣ无血清条件培养液经ＰＭ１０超滤浓缩器（分子筛）分离，获得分子量＜１０ｋＤ（１ｄａｌｔｏｎ＝０．９９２１ｕ）蛋白质滤过液及＞１０ｋＤ蛋白质浓缩液，分别加入２４孔、９６孔培养板内脊髓前角神经元（ＳＡＨＮ）的培养液中，用倒置显微镜及ＭＴＴ微量自动比色法进行神经营养活性观察。结果：在＞１０ｋＤ蛋白分子影响下，ＳＡＨＮ在体外培养４８小时仍存活良好，并伴有少部分ＳＡＨＮ长出短突起。ＭＴＴ微量自动比色法进一步显示，＞１０ｋＤ蛋白分子神经营养活性在统计学上显著高于＜１０ｋＤ蛋白分子。结论：成年大鼠瓦勒变性期ＳＣ在无血清培养条件下仍能合成与分泌神经营养因子，其活性存在于＞１０ｋＤ蛋白分子中。

Objective:To explore the promoting peripheral nerve regenerating substances from Schwann cell (SC) of adult rat sciatic nerve undergoing Wallerian degeneration.Methods:215 ml of prepared SC-serum free conditioned medium (SC-SFCM) were concentrated and dialyzed in a ultrafiltration unit with Amicon PM10 membrane (a molecular sieve),recovering >10kD concentrated fraction and <10kD filtrated fraction,which were respectively added into the mediums in 24-well and 96-well plates plated by spinal anterior horn neuron (SAHN). The neurotrophic activities from proteins of SC-SFCM were observed by phase contrast microscope and MTT assay.Results:Cultured SAHN survived well in vitro 48 hours under influence of >10kD fraction,small number of SAHN was growing with short neurites.MTT assay further showed that neurotrophic activities from >10kD fraction were significantly higher than < 10kD fraction in statistics.Conclusions:Adult rat SC undergoing Wallerian degeneration can still synthesize and secrete neurotrophic factors under serum free cultivation,the activities of which existed in >10kD fraction.