归芪多糖的制备及其延缓衰老的作用机制研究

With the improvement of people's living standard and the coming of aging society, the transformation of people health concept and medical model, their quality of life and healthy demand have increasingly improved. How to prevent disease and aging, to seek non-toxic, no side effects and effective anti-aging drug has become a hot issue in the field of medicine study..The purpose of this work was to examine whether Guiqi polysaccharides has a synergetic anti-aging effect through hemopoiesis and invigorate the circulation of blood of angelica polysaccharide, and antioxidation and improve immune function astragalus polysaccharides. High purity Guiqi polysaccharides were prepared with water extraction and alcohol sink, and DEAE cellulose column and glucan gel methods. The aging mice were used as the research object, the mitochondrial DNA of the brain, kidney and skin tissues, the expression of Fas, FasL, BCL-2 and Bax in T cells, the changes of WBC、RBC、PLT and BMNC, the expression of Sca-1 + BMNC CD44 and CD49, BMNC cell cycle changes, bone marrow cell proliferation activity were respectively assayed to study Guiqi polysaccharides' influence on the aging mice in vivo. The HUVECs、WI-38 and M-07 cell were used as the study targets, the expression of protein P16 and p53, the influence of the telomere length and telomerase activity and the M-07 cell proliferation were respectively determined to clarify the anti-aging mechanism of Guiqi polysaccharide in vitro.These reveals of mechanisms will provide an new scientific basis for the development of anti-aging medicine.

随着人民生活水平的提高和老龄化社会的到来，人们的健康观念的转变，对于生活质量和健康水平的需求日趋提高，如何预防疾病和延缓衰老，寻求既无毒副作用又方便有效延缓衰老药物的研究已成为当前医药学研究领域中的热点问题。本项目拟以当归和黄芪为研究对象，制备出高纯度的归芪多糖。然后以衰老小鼠为研究对象，研究归芪多糖对脑、肾和皮肤组织中线粒体DNA、对凋亡相关蛋白Fas、FasL、bcl-2及Bax在T细胞上的表达、对外周血及骨髓单个核细胞、对Sca- 1+BMNC 粘附分子CD44 和CD49d 的表达、对BMNC细胞周期以及骨髓细胞增殖活性的影响。再分别以人HUVECs、WI-38细胞和M-07e为研究对象，研究归芪多糖对衰老相关蛋白p53和P16的表达；对端粒长度和端粒酶活性以及对M-07e细胞增殖的影响，揭示归芪多糖在体内外延缓衰老的作用机制。这一机制的揭示将为抗衰老药物的开发提供新的科学依据。

随着人民生活水平的提高和老龄化社会的到来，人们对健康水平和生活质量的需求日趋提高，如何预防老年性疾病和延缓衰老，寻求既无毒副作用又方便有效延缓衰老药物的研究已成为当前医药学研究领域中的热点。 .本项目依据金•李东垣•《内外伤辨惑论》卷之《暑伤胃气论》中的当归补血汤古方，以甘肃产道地药材当归和黄芪为主要原料，按照一定的比例从中提取、分离和纯化出归芪多糖，系统分析其理化性质及结构，研究其体外和体内抗衰老作用及机理。首先，通过物理、化学和光谱等方法明确了归芪多糖AAP-2A是一种新的中性杂多糖，其总糖含量为 98.98%，相对分子质量大于 6.68×105 Dol，绝对分子质量为 2.252×106 Dol，主链骨架由 1,3-α-L-鼠李糖，1,3-β-D-半乳糖，1,3、1,5、1,3,5-α-L-阿拉伯糖和 1,4、1,4,6-α-D-葡萄糖组成，侧链分支点位于1,3,5-α-L-阿拉伯糖的 3位和 1,4,6-α-D-葡萄糖的 4 位，侧链分支由 1 位连接的α-D-葡萄糖组成。其次，在体外环境下，以WI-38，HUVECs和M-07e为研究对象，采用MTT法、ELISA法、Southern blot法、流式细胞术以及PCR技术从不同角度全方位发现归芪多糖通过降低衰老细胞SA-β-半乳糖苷酶活性，增强端粒酶活性和端粒长度、加快G1期的细胞进入分裂期，下调p53和p16蛋白表达，降低人巨核细胞M-07e细胞Caspase-3和Bax的表达延缓了细胞的衰老。最后，从总体水平，通过衰老大鼠的重要脏器和造血机能，进一步探究到归芪多糖可通过降低脑组织p16蛋白表达，提高脑组织端粒酶活性和脑线粒体抗氧化能力，降低肾组织线粒体DNA的含量和线粒体的缺失，提高皮肤组织SOD的活力和HYP含量，降低其MDA含量起到保护大鼠脑组织衰老，促进肾脏线粒体结构和功能的恢复，达到延缓机体衰老的作用。同时，归芪多糖还可下调衰老大鼠T淋巴细胞中Fas和FasL蛋白的表达，降低大鼠骨髓Sca-1+BMNC的粘附分子CD44和CD49d的表达，从而阻止造血细胞对骨髓基质的粘着力而使其“外移”至外周血，升高外周全血细胞，间接反映归芪多糖良好的造血功能的同时，进一步揭示出归芪多糖延缓大鼠衰老的作用机理，为植物多糖的开发和延缓衰老药物的研制提供了科学依据。