【作者】 谢云；

【导师】 饶平凡；

【作者基本信息】 福州大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 为了阐明鹿茸的生理功效的生化机理，本课题运用Sephacryl S-200HR凝胶色谱、SOURCE 15Q阴离子交换色谱、C18反相色谱和Superdex peptide 10／300GL高效液相色谱等分离手段，以相应的细胞增殖活性为指标，从冷冻干燥鹿茸粉抽提液中分离纯化出两种具有生物活性的物质：P2-3和b组分。 P2-3组分在SDS-PAGE和PAGE两类电泳图上均显示一条带，说明该组分已经达到电泳纯，通过标定得到其分子质量为53.3kDa，同时利用离子交换层析法测定其等电点pI为5.4。经活性鉴定发现，该蛋白不仅具有促进肾上皮细胞增殖的活性，同时还可促进小鼠脾细胞的生长，而且两种细胞的增殖的趋势相类似：细胞的增殖作用先随着P2-3组分浓度的增大而增大，当蛋白含量超过一定浓度时，细胞的增殖作用会有所下降。蛋白含量达到0.124mg／ml时，两种细胞的增殖效果均达到最大，其中，MDCK的增殖率达73.0％，脾细胞的增殖率达63.3％。通过比较发现，该组分促MDCK细胞的增殖效果要比脾细胞的增殖效果好。 b组分经毛细管液相色谱鉴定为单一组分，利用凝胶色谱法测定其分子质量，得到其分子质量的值为478 Da。该组分在低蛋白浓度下对MDCK细胞的增殖有抑制作用，此后细胞的增殖效果会随着蛋白浓度的增加而增加，但当蛋白超过一定浓度时，增殖作用亦会有所下降，其中以蛋白浓度为11.1μg／ml时的作用效果最佳，增殖率达64.3％。b组分对脾细胞亦有促增殖作用，但增殖效果没有MDCK增殖效果显著，其最佳作用的蛋白浓度也是在11.1μg／ml，但增殖率仅达34.9％。 综上所述，鹿茸中确实含有促进上皮细胞增殖的活性组分，而细胞的增殖与分化是创伤修复过程中最重要的细胞活动之一，因此，该研究的发现有可能为创伤修复提供分子生物学机理以及为筛选鹿茸中创伤愈合药物提供基础数据。同时本研究还发现鹿茸对小鼠脾细胞亦具有促进增殖的作用，因脾细胞中含有多种免疫细胞，在机体的免疫系统中发挥着重要作用，故该发现还有可能为鹿茸增强免疫的生理功效的研究提供有利的线索。更多还原

【Abstract】 In order to elucidate the biochemical mechanism of therapeutic effects of pilose antler, the extraction of freeze dried pilose antler powder was fractionated by a combination of chromatography of different chemistries such as gel filtration chromatography on Sephacryl S-200HR, ion-exchange chromatography on SOURCE 15Q, reverse phase chromatography on C18 and chromatography on Superdex peptide 10/300GL. As a result two bioactive components, P2-3 and b were obtained.P2-3, electrophoretically pure, with a molecular of 53.3 kDa, were demonstrated to be capable of increasing the proliferation rate of both Madin-Darby canine kidney cells (MDCK) and mice spleen cells, with a similar function pattern. With the increase of P2-3 concentration, the proliferation rate of MDCK cells increased at first, and then decreased when the concentration of P2-3 exceeded a certain value, with the maximum of 73.0% and 63.3% increase for MDCK cells and spleen cells, respectively at the concentration of 0.124mg P2-3/ml. The isoelectric point of P2-3 fraction is 5.4, as was measured by ion-exchange chromatography.b fraction, which was proved to be pure by capillary reverse phase chromatography, was demonstrated to also promote the proliferation of both MDCK cells and mice spleen cells. MDCK cell proliferation was inhibited at low concentration. With the increase in its concentration, MDCK cell proliferation was enhanced to reach the maximum of 64.3% of the control group at a concentration of 11.1 μg/ml. The effect of b fraction on mice spleen cell proliferation was less potent than on MDCK cells, with the maximum of 34.9% at 11.1μg/ml concentration.Since cell proliferation and differentiation constitute a major process in wound healing, the isolation P2-3 and b from pilose antler and the identification of their bioactivity of increasing the proliferation of epithelial cells, have provided another molecular clue to the elucidation of pilose antler’s therapeutic effect in wound healing, as well as to the development of a new wound healing drugs from pilose antler. Consisting of various immune cells, spleen plays an important role in the immune更多还原