梅花鹿鹿茸中促PC12细胞增殖蛋白的分离和活性研究

Isolation and PC12 cell proliferative protein fraction from pilose antler and its activity

目的对冻干鹿茸水溶性组分进行分离,并考察鹿茸水溶性组分及其分离组分对PC12细胞的促增殖活性。方法应用S-200分子筛凝胶液相色谱和DEAE阴离子交换液相色谱分离鹿茸水溶性组分,进行蛋白质SDS-PAGE电泳分析,Folin-酚法测定鹿茸样品中的蛋白,并用MTT法测定PC12细胞增殖率。结果鹿茸水溶性组分在13.3 mg/mL能促进PC12细胞增殖47%(P<0.01),其蛋白质量分数为84.2%;经分子筛色谱分离的S200-P2组分具显著促PC12细胞增殖活性(46.0%,97.0μg/mL,P<0.01);经离子交换色谱进一步分离的DEAE-P1组分在51.0μg/mL下的PC12细胞的增殖率为111.5%(P<0.001)。DEAE-P1组分的蛋白主要集中分布于Mr56 000和大于Mr 100 000处。结论冻干鹿茸的水溶性组分及其液相色谱分离的一个大分子蛋白组分具有显著的促PC12细胞增殖活性,提示这个组分与鹿茸的神经生长因子样活性相关。

Objective The lyophilized pilose antler water extract （PAE） was isolated , and their cell proliferation on PC12 cells was observed. Methods S-200 Size-exclusive gel and DEAE negative ion-exchange liquid chromatograph were employed to fractionate the PAE. SDS PAGE was employed to analyze the proteins composition of PAE. The protein concentration was determined by Folin-Phenol assay. The proliferation rates of PC12 cells were measured by MTT assay. Results The proliferation rate of PAE on PC12 cells at 13.3 mg/mL was 47% （P〈0.01）. PAE＇s protein content is 84.2%. The PAE was fractionated and obtained two active fractions： S200-P2 and DEAE-P1, whose cells proliferation rates were 46% （97.0 μg/mL, P〈0. 01） and 111.5% （51.0μg/mL, P〈0. 001）, respectively. DEAE-P1 mainly had proteins of Mr 56 000 and higher than Mr 100 000 molecular weight. Conclusion The cell proliferation of PAE and its chromatographic fraction indicate that this fraction may attribute to pilose antler＇s never growth factor-like activity.