蜂王浆主蛋白的抗衰老作用及分子机理研究

Royal jelly is one of honey bee products which has been globally considered to possesses anti-aging function for animal and human. It was reported recently that the main royal jelly proteins (MRJPs) are the key factors in royal jelly which induce queen differentiation in honeybee. However, whether MRJPs possess ant-aging function to human cells and mammal animal still remained unclear untill now. Based on previous solid researches, this project will observe its effects on the lifespan of wild strain and gene mutants in Drosophila, explore its effects on the cell proliferation activity, replicative and changes of cell senescence of human embryonic lung diploid fibroblasts, and investigate its effects on the aging of organ and tissues of senescence model rat. Then, the biochemical difference of the activity of antioxidases and the content of malonyldialdehy between treatments and controls will be determined. Through analysis of gene expression differences of different samples, the differential genes will be screened with genechiop detection. The high expression genes and proteins associated with senescence of these tests models will be used to analyze the differences of gene expression and protein level between tratments and controls with real time PCR reaction and Western blot. The differences of telomerase of different samples will be detected as well. All these data will be analyzed with the methods of bioinformatics and signal pathway. These results will reveal the anti-aging molecular mechanisms of MRJPs on the model organisms and mammal animal, and provide new important knowledge for firstly explore the functional food products of MRJPs in the world.

蜂王浆是国际公认的对动物和人体具有抗衰老功能的蜂产品。新近发现王浆主蛋白MRJPs是蜜蜂幼虫发育成蜂王的决定性因素，但关于MRJPs对人体细胞和哺乳动物的抗衰老功能尚不明确。本课题将在现有扎实研究的基础上，作MRJPs对野生和基因突变株果蝇寿命的影响，对人胚肺成纤维细胞传代次数、生长和衰老的影响，对衰老模型大鼠器官组织衰老的影响研究。然后检测MRJPs对受试与对照样本的超氧化物歧化酶活性，对丙二醛等过氧化物含量等生化指标的影响；用基因芯片检测受试与对照样本的基因表达谱差异，筛选出差异基因；选择衰老高表达基因或蛋白，用实时荧光PCR和Western blot检测受试与对照样本的基因表达谱与蛋白质水平差异。并用实时荧光PCR测定不同样本的端粒酶活性差异，最后作生物信息和信号通路分析。结果将揭示MRJPs对模式生物和哺乳动物的抗衰老分子机理，为在国际上率先开发MRJPs抗衰老功能食品提供科学依据

蜂王浆是国际公认的对动物和人体具有抗衰老功能的蜂产品。王浆酸一直被作为蜂王浆的主要生物活性成分。近年研究证实，王浆主蛋白（MRJPs）是王浆的关键活性成分，但对其功能缺乏系统证据。.本课题采用从蜂王浆中超滤得到MRJPs冻干粉作果蝇和老年大鼠干预，人肺胚纤维细胞HFL-I培养，观察MRJPs对这三种模型的抗衰老作用。果蝇试验显示，饲料中添加1.25%、2.50%、5.00% MRJPs使雄果蝇平均寿命分别延长4.3%、9.0%、12.4%和13.9%，使雌果蝇平均寿命分别延长5.8%、9.7%、20.0%和11.8%，MRJPs有提高果蝇摄食量和繁殖力作用，提高果蝇SOD(Cu, Zn)活力，降低脂质过氧化产物丙二醛（MDA）含量，促使SOD(Cu, Zn)、Egfr、MAPK和S6K上调。培养基中添加0.1、0.2、0.3 mg/mL MRJPs培养HFL-I细胞，显示MRJPs具有促细胞增殖作用，延长细胞进入复制型终末时间。细胞形态、细胞群加倍数、衰老细胞染色观察显示，MRJPs能够延缓细胞衰老。RT-PCR和拉曼光谱检测显示，MRJPs具有保护端粒，促进DNA及蛋白质在HFL-I细胞中的合成作用。对老年大鼠补充MRJP 125 mg/Kg.d和酪蛋白125 mg/Kg. d，持续14周后水迷宫法观察显示，补充MRJPs能改善大鼠的空间认知能力。尿液代谢组学分析结果显示，MRJPs干预后促使大鼠呈回归青年期趋势。器官组织切片显示，MRJPs可改善由衰老引起的肝脏及肾脏的病理性特征。生化检测结果，MRJPs能提高大鼠血液过氧化氢酶（CAT）活力，降低MDA水平，显示抗氧化活性。RT-PCR检测显示，MRJPs干预能提高抗衰老相关基因foxo1基因转录水平，上调mTOR和 S6K 基因转录水平，显著提高血液中端粒酶逆转录酶TERT基因的表达。结论：通过补充膳食MRJPs可以促进机体抗氧化能力的提高及消除自由基活性，细胞增殖、改善空间认知能力，衰老相关基因的下调，保护端粒酶，促进DNA及蛋白质的合成，从而产生延长寿命的效果。此课题研究从动物和细胞分子水平证明了MRJPs为蜂王浆中具有抗衰老作用的关键生物活性物质，为蜂王浆的质量控制和评价、标准制订提供了重要科学依据，将促进蜂王浆深加工、王浆蛋白新产品开发和市场推广。