神经再生条件液中相对分子质量为22万的蛋白提取与鉴定

EXTRACTION AND IDENTIFICATION OF THE PROTIEN BAND OF 220 000 IN NERVE REGENERATION CONDITIONED FLUID

目的了解神经再生条件液（never regeneration conditioned fluid,NRCF）中蛋白质成分组成及相对分子质量为220×103的蛋白带中是否存在一类新的尚未知的神经活性因子。方法新西兰大白兔25只,体重1.8～2.5kg,取坐骨神经建立神经再生室模型。术后1周采集NRCF,采用凝胶过滤法分离NRCF中蛋白质,主要分离相对分子质量为220×10^3的蛋白带,应用自然聚丙烯酰胺凝胶电泳（native-polyacrylamide gel electrophoresis,Native-PAGE）法检测其相对分子质量。并对取得的相对分子质量为220×10^3（a峰）和（20～40）×10^3（c峰）的蛋白带,分别应用已知的神经活性因子抗体：抗神经生长因子（nerve growth factor,NGF）抗体、抗胶质细胞源性神经营养因子（glial cell-derived neurotrophic factor,GDNF）抗体、抗脑源神经营养因子（brain-derived neurotrophic factor,BDNF）抗体、抗神经营养素3（neurotrophin 3,NT-3）抗体、抗NT-4抗体、抗睫状神经营养因子（ciliang neurotrophic factor,CNTF）抗体,采用Western blot免疫印迹法和ELISA检测,观察抗原抗体反应。结果经凝胶过滤法分离的NRCF蛋白质,多集中在相对分子质量为（20～40）×10^3和220×10^3。相对分子质量为（20～40）×10^3蛋白带中神经营养因子与抗NGF、抗BDNF、抗NT-3、抗CNTF等抗体反应,相对分子质量为220×10^3的蛋白带所含神经营养因子仅与抗NT-4抗体反应。结论NRCF中相对分子质量为220×10^3的蛋白带仅与抗NT-4抗体反应,且生物活性远强于NT-4,该蛋白带中可能存在一类新的神经活性因子。

Objective To separate each protein band from the nerve regeneration conditioned fluid（NRCF）and to study whether there are some new and unknown neurotrophic factors in the protein bands with a relative molecular mass of 220×10^3. Methods The silicone nerve regeneration chambers were formed in the sciatic nerve of the 25 New Zealand rabbits （weight, 1.8-2.5 kg）, and NRCF was taken from it at 1 week after operation. The Nativepolyacrylamide gel electrophoresis （Native-PAGE） was used for separating the proteins from NRCF and detecting the relative molecular mass. The Western blot and ELISA were used to observe whether the protein bands [220×10^3（Band a ）, （20-40）×10^3（Band c）] of NRCF could combine with the antibody of the known antibody of neurotrophic factor （NTF） ：nerve growth factor （NGF）, glial cell-derived neurotrophic factor （GDNF）, brain-derived neurotrophic factor （BDNF）, neurotrophin 3（NT-3）, NT-4, ciliang neurotrophic factor（CNTF）. Results Separated by Native-PAGE, NRCF mainly contained two protein bands：Band a had a relative molecular mass about 220 ×10^3, and Band c had a relative molecular mass about （20-40）× 10^3. Band a could not combine with the antibodies of the NGF, BDNF, CNTF, and NT-3, but could combine with the antibody of NT-4. Band c could combine with the antibodies of NGF, BDNF, CNTF and NT-3, but could not combine with the antibodies of NT-4 and GDNF. Conclusion The protein bands with a relative molecular mass of 220×10^3 have stronger neurotropie and neurotrophic effects than the protein bands with a relative molecular mass of （20-40）×10^3, which contains NGF,CNTF, etc. NT-4 just has a weak or no effect on the sympathetic neurone. This indicates that there is a new NTF in the protein bands with a relative molecular mass of 220×10^3, which only combines with the antibody of NT-4.