覆盆子中糖蛋白的分离纯化、抗衰老活性及其对klotho基因的表达调控研究

To research the senescence mechanism is always a focus in geriatric medical and biological study, and to reveal the mechanism could delay the aging speed and extend the life fundamentally. Traditional Chinese Medicine (TCM) holds that the kidney is the congenital foundation, and the kidney deficiency is the basic reason of senescence, therefore, to tonify kidney is the basic means of anti-aging. Therefore, it is very significant for the development of new anti-aging drugs to study on the anti-aging activities and mechanism of Traditional Chinese Medicine with tonifying kidney. As an important medicine of the ancient anti-aging compounds, Fupenzi (Rubus chingii Hu.) has a concrete manifestation of anti-aging effects on its replenishing the kidney and invigorating Yang. However, the anti-aging mechanism of Fupenzi remains unclear so far. Modern pharmacological research showed that the aging was closely concerned with the accumulation of free radicals and their own gene. Klotho gene is a new aging gene and is predominantly detected in the kidney and cerebullar choroids. With the function of Fupenzi and the role of klotho gene in anti-aging, in this study the glycoprotein with efficient scavenging the free radicals are regarded as anti-aging materials in Fupenzi, and the anti-aging activities of them are evaluated systematically in vitro and in vivo, furthermore, their effects on the modulation mechanism of anti-aging klotho gene expression in rat kidney tissue are also investigated. The results obtained from this study will synthetically evaluate the anti-aging material foundation and reveal the anti-aging mechanism of Fupenzi, and the results will elucidate the anti-aging effect of Fupenzi scientifically.

衰老机理的研究一直是老年医学和生物学研究的前沿，揭示衰老机理可望从根本上推迟衰老速度，在延长寿命方面获得突破。中医理论认为肾虚是人体衰老的根本原因，补肾是抗衰老的基本途径。因此，研究补肾中药的抗衰老活性及抗衰老机理，对于开发新的抗衰老药物具有非常重要的指导意义。覆盆子具有“益肾、固精、缩尿”之功效，是抗衰老作用的具体体现，并且千百年来一直就是抗衰延寿方中的重要一员。然而，有关于覆盆子的抗衰老机理却缺乏相应的研究。研究表明，衰老与机体中自由基的积累和本身的基因密切相关。Klotho基因是近年在肾脏组织中发现的又一个与衰老相关的基因。结合覆盆子延缓衰老的功效及klotho基因在抗衰老方面的重要作用，本项目以覆盆子中具有清除自由基的糖蛋白成分为研究对象，系统研究糖蛋白成分的体内外抗衰老活性及对小鼠肾组织中klotho基因表达的调控作用，使覆盆子抗衰老作用得到现代意义的阐明。

采用水溶醇沉法提取覆盆子的总糖蛋白，通过DEAE-52和Sephadex G-100柱层析分离，共获得4个单一的糖蛋白分子。SDS-PAGE电泳和多糖染色表明，4个糖蛋白分子纯度较高，且分子量均约为20.1 kDa。体外抗氧化实验及细胞学实验研究表明，3号糖蛋白分子的抗氧化性及抗衰老活性最强，而体内研究也表明，3号糖蛋白分子能有效改善衰老小鼠肾组织器官病理状态，使肾储备能力得到修复，从而起到补肾的作用，并且能明显改善衰老小鼠体内总超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶及丙二醛的生理指标，具有明显的抗衰老活性。通过RT-PCR、q-PCR和Western blot方法对小鼠肾组织中的klotho基因表达进行检测，结果表明3号糖蛋白能明显增加小鼠肾组织中抗衰老基因klotho的表达量。因此推测覆盆子抗衰老的机制可能是通过清除体内有害的自由基，从而改善机体器官的生理状态，使各组织器官的功能得到修复，特别是对肾组织的修复可能与抗衰老作用直接相关。此外，覆盆子的抗衰老作用也可能通过调节体内抗衰老基因klotho的表达水平而实现其抗衰老作用，这一结果为覆盆子作为传统抗衰延寿方中的重要一员提供科学依据，同时也为“补肾是抗衰老的基本途径”这一中医观点提供实验基础。项目资助发表SCI论文4篇，待发表SCI论文1篇，培养硕士研究生3名，其中2名已获得硕士学位，1名在读。