本文關(guān)鍵詞：王漿主蛋白的抗衰老功能及分子機(jī)理研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蜂王漿 王漿主蛋白 抗衰老 抗氧化 空間記憶 骨密度 代謝組學(xué)

【摘要】：隨著國內(nèi)外人口老齡化問題的加劇,國際市場對抗衰老功能食品的需求越來越大,蜂王漿(RJ)作為國際公認(rèn)的傳統(tǒng)抗衰老功能食品,將發(fā)揮更加重要的作用。根據(jù)新近研究報(bào)道,王漿主蛋白(MRJPs)是RJ的關(guān)鍵活性成分,但是否對哺乳動物具有抗衰老作用及作用機(jī)理尚未見報(bào)道。本研究以從新鮮RJ超濾分離的MRJPs凍干粉為研究對象,以自然衰老大鼠和人體細(xì)胞為模型,進(jìn)行了 MRJPs的抗衰老作用及分子機(jī)理系統(tǒng)研究,結(jié)果如下:1.MRJPs對大鼠的抗衰老作用及分子機(jī)理電泳結(jié)果顯示MRJPs集中在49-80 kDa,其氨基酸組成與酪蛋白相似,說明MRJPs的功能不是體現(xiàn)在氨基酸水平上,而是通過特殊的形式來表現(xiàn)其生物活性。衰老大鼠的附睪和睪丸指數(shù)顯著低于青年大鼠,口服MRJPs并不能改變這種臟器的退化,對其它臟器指數(shù)和體重也未產(chǎn)生明顯影響。蘇木精-伊紅染色結(jié)果表明,MRJPs可以明顯改善衰老引起的大鼠肝臟及腎臟等重要器官出現(xiàn)的病理性特征。口服MRJPs能顯著提高大鼠血液的CAT活力(P0.05),降低血液的脂質(zhì)過氧化物MDA水平;MRJPs干預(yù)組的GSH含量與GSH-PX活力與青年大鼠表現(xiàn)出類似的調(diào)節(jié)方式,顯示MRJPs具有良好的抗氧化活性。mTOR轉(zhuǎn)錄水平與MRJPs干預(yù)劑量呈正相關(guān)。S6激酶(S6K)通過mTOR徑被激活,并出現(xiàn)轉(zhuǎn)錄水平上調(diào)趨勢。青年大鼠的mTOR基因轉(zhuǎn)錄水平顯著高于自然衰老對照組,而MRJPs干預(yù)能提高衰老大鼠的mTOR基因轉(zhuǎn)錄水平,類似現(xiàn)象在s6k基因中也有發(fā)現(xiàn)。青年大鼠的Foxo1基因轉(zhuǎn)錄水平顯著高于自然衰老對照組,而MRJPs干預(yù)能提高衰老大鼠Foxo1基因轉(zhuǎn)錄水平,顯示抗衰老活性。此外,MRJPs能顯著提高大鼠血液中端粒酶逆轉(zhuǎn)錄酶TERT基因的表達(dá),說明其抗衰老機(jī)理可能與TERT的轉(zhuǎn)錄水平的調(diào)節(jié)有關(guān)。與SOD未出現(xiàn)明顯改變一樣,Sod1因的轉(zhuǎn)錄水平在干預(yù)后也未出現(xiàn)明顯變化。2.MRJPs對大鼠認(rèn)知功能的改善作用動物水迷宮實(shí)驗(yàn)表明,逃避潛伏期較對照和酪蛋白組分別縮短了 48.5%(P=0.0045)和49.9%(P=0.0036);穿過目標(biāo)區(qū)域的次數(shù)較對照和酪蛋白組分別提高了 177.4%和46.1%,說明MRJPs能顯著改善老年大鼠的空間認(rèn)知能力�；诔咝б合嗌V四極桿飛行時(shí)間質(zhì)譜(UPLC-QTOF/MS)的大鼠尿液指紋圖譜表明,MRJPs干預(yù)衰老大鼠后,其代謝特征出現(xiàn)回歸青年大鼠的趨勢。鑒定了 28個(gè)與衰老相關(guān)的生物標(biāo)記物,其中6個(gè)標(biāo)記物,煙酸單核苷酸(1),丙氨酸丁氨酸硒醚(2),CDP-乙醇胺(3),磺基丙氨酸(4),磷酸烯醇式丙酮酸(5)和黃嘌呤核苷(6)在MRJPs干預(yù)后發(fā)生顯著改變。結(jié)合文獻(xiàn)RJ改善記憶的功能分析,RJ來源的MRJPs有可能是一種新的提高認(rèn)知功能的補(bǔ)充蛋白質(zhì)。3.MRJPs及KHC03對大鼠骨密度的改善作用補(bǔ)充MRJPs及KHCO3不會影響大鼠體重。血液肝功能檢測結(jié)果表明,MRJPs及KHCO3作為膳食補(bǔ)充劑是安全的,且對肝功能有一定調(diào)節(jié)作用。MRJPs會導(dǎo)致大鼠骨密度輕微降低,推測其通過降低雌激素水平、促進(jìn)鈣流失而引起骨密度的降低。同時(shí)補(bǔ)充KHCO3(100 mg/mL)可防止骨密度的降低,并引起雌二醇水平降低,而MRJPs與雌二醇共同作用于成骨細(xì)胞時(shí)存在拮抗作用,說明MRJPs與KHC03對骨密度的聯(lián)合保護(hù)作用可能是通過降低MRJPs和雌二醇對成骨細(xì)胞的拮抗作用實(shí)現(xiàn)的。MRJPs與KHCO3的協(xié)同作用具有一定的抗氧化性。補(bǔ)充MRJPs和KHC03在一定程度上能降低CTX-Ⅰ,顯著提高Ⅰ型原骨膠原(P1NP)和OPG含量,而補(bǔ)充的KHCO3有類似的效果。MRJPs和雌雌醇能上調(diào)clock、cry1和cry2,下調(diào)bmal1,說明通過時(shí)鐘節(jié)律調(diào)控途徑可影響骨密度。4.MRJPs對MRC-5細(xì)胞的抗衰老作用當(dāng)MRC-5細(xì)胞處于中年期時(shí),補(bǔ)充終濃度25-200 μg/mL的MRJPs對MRC-5雖有一定的促進(jìn)作用,但不顯著。流式細(xì)胞檢測結(jié)果表明,MRJPs對處于中年期MRC-5細(xì)胞增殖指數(shù)的影響并不明顯。但當(dāng)MRC-5細(xì)胞進(jìn)入衰老期后,細(xì)胞增殖速率顯著降低,補(bǔ)充MRJPs具有明顯的促增殖作用。β-半乳糖苷酶活性檢測表明,補(bǔ)充MRJPs能顯著降低MRC-5衰老細(xì)胞比例,且呈劑量相關(guān)性。拉曼檢測結(jié)果顯示,除683 cm-1處外,MRJPs及酪蛋白的拉曼特征峰與對照組高度一致,體現(xiàn)其作為蛋白的共性。
[Abstract]:With the problem of population aging intensifies, the international market demand for anti-aging function food is more and more big, royal jelly (RJ) as a traditional anti-aging function internationally recognized food, will play a more important role. According to recent research reports, royal jelly proteins (MRJPs) is a key active component of RJ, but whether or not has anti-aging effect and mechanism of mammals has not been reported. In this study, isolated from fresh RJ ultrafiltration MRJPs freeze-dried powder as the research object, with the natural aging rats and human cells as a model of the system and the molecular mechanism of MRJPs anti-aging results are as follows: 1.MRJPs on rats and molecular rejuvenation the mechanism of the electrophoresis results showed that MRJPs concentration in 49-80 kDa, and the amino acid composition of casein is similar to that of MRJPs, the function is not reflected in the amino acid level, but through a special form. The biological activity of aging rats testis and epididymis index was significantly lower than that of young rats, oral administration of MRJPs did not change the degradation effect of organs, also did not produce other organ index and weight. Hematoxylin eosin staining results showed that MRJPs can significantly improve the pathological characteristics caused by the decline of old rat liver and the kidney and other important organs. The oral administration of MRJPs can significantly improve the rat blood CAT activity (P0.05), lower blood lipid peroxide levels of MDA; GSH content and GSH-PX activity in young rats and MRJPs intervention group showed a similar regulation, showed that MRJPs had antioxidant activity of.MTOR kinase and MRJPs transcription level intervention dose.S6 good (S6K) by mTOR diameter is activated, and up-regulated transcription. The transcription level of mTOR gene in young rats was significantly higher than that of the natural aging control group, MRJPs The intervention can improve the transcription level of mTOR gene in aging rats, a similar phenomenon is also found in the S6K gene. Foxo1 gene transcription level of young rats was significantly higher than that of the natural aging control group, and MRJPs intervention can improve the transcription level of Foxo1 gene in aging rats, showed anti-aging activity. In addition, MRJPs could significantly increase the expression of gene in the blood of human telomerase reverse transcriptase TERT rats, indicating its anti-aging mechanism may be related to the regulation on the transcription level of TERT and SOD. There was no obvious change, because the transcription level of Sod1 after the intervention did not appear that improve animal change significantly in.2.MRJPs water maze test on cognitive function in rats, shortened 48.5% the escape latency compared with the control group respectively and casein (P=0.0045) and 49.9% (P=0.0036); 177.4% and 46.1% times the increase through the target area compared with the control group and casein respectively, indicating that MRJPs can display To improve the spatial cognitive ability of aged rats. Quadrupole time-of-flight mass spectrometry based on ultra high performance liquid chromatography (UPLC-QTOF/MS) showed that the rat urine fingerprints, MRJPs intervention rats, the metabolic characteristics of young rats appeared regression trend. 28 old biomarkers associated with the decline of the identification. 6 markers, nicotinic acid mononucleotide (1), selenocystathionine (2), CDP- (3) ethanolamine, cysteic acid (4), phosphoenolpyruvate (5) and xanthine nucleotide (6) changed obviously in MRJPs after intervention. Literature analysis improve the memory function with RJ RJ, source of MRJPs may improve the effect of protein.3.MRJPs and KHC03 a new improved cognitive function on bone density in rats of MRJPs and KHCO3 will not affect the body weight of rats. Blood liver function test results show that the MRJPs and KHCO3 as dietary supplements are safe , and there is a certain regulatory role of.MRJPs will lead to bone mineral density in rats decreased slightly on liver function, presumably by reducing estrogen levels, promote calcium loss caused by reduced bone density. At the same time to add KHCO3 (100 mg/mL) can prevent the loss of bone density, and induced by estradiol level decreased, and MRJPs together with the female glycol in osteoblasts when there is antagonism, indicating the protective effect of MRJPs and KHC03 on bone mineral density may be through the synergistic effect of reducing MRJPs and estradiol on.MRJPs and KHCO3 realize the antagonism of the osteoblast has some antioxidant activity. MRJPs and KHC03 can reduce CTX- 1 to a certain extent, significant improve the type I procollagen (P1NP) and the content of OPG, and added KHCO3 have the similar effect of.MRJPs and estrogen estradiol can increase clock, cry1 and CRY2, down BMAL1, the clock rhythm control way can affect bone The density of.4.MRJPs on MRC-5 cell anti-aging effect when MRC-5 cell is in the middle period, adding a final concentration of 25-200 g/mL MRJPs of MRC-5 has a certain role in promoting, but not significant. Flow cytometry results showed that the effect of MRJPs on MRC-5 cell proliferation index is in the middle period is not obvious. But when cells enter MRC-5 after the aging period, the cell proliferation rate was significantly reduced, MRJPs has significantly stimulated the proliferation. Detection indicated that beta galactosidase activity, MRJPs can significantly reduce the aging cell ratio of MRC-5, in a dose-dependent manner. The Raman detection results show that the addition of 683 cm-1, MRJPs and Raman spectra of casein and control group of highly consistent, embodied as a common protein.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：TS201.21

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