【作者】 原江水；

【导师】 张士璀；

【作者基本信息】 中国海洋大学 ， 发育生物学， 2013， 博士

【摘要】 在有机体的衰老过程中，免疫功能出现下降的趋势，特异性和非特异性免疫应答均出现相应的变化。免疫老化是一个相当复杂的过程，已有研究表明人类寿命与机体的免疫功能密切相关，免疫系统是机体保护自己免受其他物质损害的对体内外环境的适应和反应体系。同时，免疫系统在细胞和分子水平上与细胞分化发育、肿瘤、自身免疫性疾病等过程密切相关，从根本上参与了机体老化的整个过程。白藜芦醇（Resveratrol, RSV）属于非黄酮类多酚化合物，是许多种子植物包括葡萄和浆果在遇到不利环境中所产生的一种天然的植物抗毒素，实验证据表明白藜芦醇具有抗衰老、抗肿瘤、抗炎症和免疫调节的作用，可以提高老龄小鼠和人的免疫功能。尽管白藜芦醇长久以来因其促进健康，抵抗衰老和增强免疫的特性而受到高度评价，但是其调节免疫功能还缺乏系统的科学证据，其增强免疫功能的内在机制不甚明确。本研究拟在于探索白藜芦醇的口服吸收对年轻大鼠、中年大鼠和老年大鼠获得性免疫及谷氨酰半胱氨酸连接酶的影响，为白藜芦醇在免疫调节和抗衰老领域的应用建立数据基础。在本研究中，我们使用全自动生化分析仪和血液分析仪测定白藜芦醇对大鼠血液学/生物化学指标的影响，探讨生理条件下白藜芦醇是否影响大鼠的血液学和生物化学指标，结果显示白藜芦醇的口服吸收对大鼠体重及红细胞、白细胞、血小板、血红蛋白、谷丙转氨酶、谷草转氨酶、高密度脂蛋白、低密度脂蛋白等血液学指标未造成明显变化。因迟发型免疫应答（DTH）会随着年龄的增加而减弱，弱DTH反应或没有DTH反应常常与疾病的发病率和死亡率的升高相关联，我们采用免疫注射的方法，探讨白藜芦醇对不同年龄段大鼠的DTH应答的影响，结果显示白藜芦醇能够显著增强老龄大鼠的DTH反应。老龄个体体液免疫应答下降的主要表现是针对许多疫苗的免疫应答、抗体滴度的产生以及抗原抗体亲和力的持续下降，为探讨白藜芦醇对抗体产生的影响，我们采用免疫注射KLH后ELISA法检测大鼠血清不同时间点抗KLH特异性IgG、IgG1和IgG2抗体的变化，探讨其对体液免疫应答的影响，结果显示白藜芦醇显著增强老龄大鼠的抗KLH特异性IgG、IgG1和IgG2抗体的产生，明显提高了老龄大鼠对抗原的反应性，增强了体液免疫应答反应。在衰老过程中，T淋巴细胞的总数量和各亚群所占的比例发生显著的变化，通过对大鼠脾脏中T细胞和B细胞组成进行分析，探讨白藜芦醇可能对细胞免疫功能产生的影响；检测口服白藜芦醇后大鼠脾细胞对T细胞有丝分裂原ConA、B细胞有丝分裂原LPS和特异性抗原KLH刺激后的增殖情况，探讨其对细胞增殖可能的影响；结果显示白藜芦醇比较明显的提高老龄大鼠CD4+T细胞的数量，提示白藜芦醇对老龄大鼠的细胞免疫应答可能起到调节的作用。谷胱甘肽（GSH）在保持细胞的氧化还原状态中发挥十分重要的作用，GSH的水平随着衰老发生的变化是衰老研究中的热点问题，谷氨酰-L-半胱氨酸连接酶(glutamyl-L-cysteineligase,GCL)，以前被称为γ谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,γ-GCS)，是GSH从头合成过程中催化酶促反应第一步反应的催化酶，也是谷胱甘肽从头合成过程中的限速酶，我们通过荧光定量PCR、Western Blot和酶活分析等方法研究大鼠心脏、脑和肝脏中GSH代谢过程中的关键酶谷氨酰半胱氨酸连接酶（GCL）催化亚基（GCLC）和调节亚基(GCLM）在不同年龄段中的变化及白藜芦醇对老龄大鼠各组织中GCL基因表达、蛋白含量和酶活性的的影响，结果显示在肝脏、心脏和脑组织中谷氨酰半胱氨酸连接酶催化亚基和调节亚基的基因表达和蛋白含量随着年龄增长，在中年组达到较高的水平，至老年组出现明显的下降，白藜芦醇能够显著提高老龄大鼠肝脏、心脏、脑中谷氨酰半胱氨酸连接酶催化亚基和调节亚基的基因表达、蛋白含量和酶活性水平。综合以上实验结果，我们认为适量的白藜芦醇口服吸收在不影响血液学指标和体重的情况下，在老龄Wistar大鼠中通过增强体液免疫应答和细胞免疫应答能够起到的免疫调节和保护作用；通过调节老龄大鼠中谷氨酰半胱氨酸连接酶的含量和酶活性提示白藜芦醇可能通过调节谷氨酰半胱氨酸连接酶进而调节GSH的含量而发挥抗衰老的效应。本实验首次采用多种技术手段和方法从多个角度研究白藜芦醇对老龄个体适应性免疫应答和衰老过程中的重要标记物GSH代谢关键酶谷氨酰半胱氨酸连接酶的影响，在一定程度上揭示了白藜芦醇的免疫调节和保护作用，为白藜芦醇在免疫功能调节和抗衰老研究中提供数据基础。更多还原

【Abstract】 Aging affects the immune system, leading to diminished overall functions. Thisphenomenon has been termed immunosenescence, which is clearly manifested byage-dependent defects in both humoral and cell-mediated immune responses.Resveratrol (3,5,40-trihydroxystilbene; RSV), a polyphenol, is a bioactive substancewith multiple functions, and occurs naturally in several plant species includinggrapevines and berries. Accumulating data have suggested that RSV hasanti-carcinogenic, anti-inflammatory, anti-microbial, anti-viral and anti-oxidantproperties that might be relevant to chronic diseases and/or longevity in humans.However, the mechanism underlying these observed anti-aging andimmunoregulation effects is poorly understood. The present study was undertaken toaddress the effect of dietary intake of resveratrol on the adaptive immunity andexpression of the glutamate cysteine ligase in aged rats.The hematologic indexes of the blood and the biochemical parameters of theserum were analyzed using flow cytometry and enzymatic analysis. Body weightswere not significantly affected by dietary RSV supplementation.The results revealedthat haemotological indice and biochemical parameters including ALT/AST activitiesand TG, TC, HDL and LDL levels of young, middle-aged and aged rats all remainedsimilar among the diet groups, suggesting that general physiology of rats was notaffected by dietary RSV supplementation.Among in vivo parameters of T cell-mediated immune response, thedelayed-type hypersensitivity (DTH) reaction is depressed with aging. Particularly,low or no DTH responses are often predictive of morbidity and mortality. The Tcell-dependent humoral response (antigen-specific antibody production) wasmeasured after immunization of rats with KLH. The response was calculated as thedifference in ear thickness of the rats before and after challenge with a recall antigen.Dietary RSV supplementation significantly affected the DTH response in aged rats.Although the B cell compartment of the immune system is only influenced to a minor extent by immuno-senescence, antigen-specific responses to vaccination arealtered with aging. The levels of KLH-specific IgG, IgG1and IgG2in the bloodwere measured by an enzyme-linked immunosorbent assay (ELISA). Levels ofanti-KLH IgG, anti-KLH IgG1and anti-KLH IgG2were both significantlyincreased in aged rats fed RSV-supplemented diet.One of the best characterized changes commonly observed in elderly subjectsand old mice is the deficiencies in T cell functions that are exemplified by decreasedT cell memory and exhaustion of the na ve T cell population with involution of thethymus. The percentages of major T and B cell subsets in spleen were assayed usingfluorescence-activated cell sorting (FACS) analysis. Proliferation of splenocytes werestimulated by ConA, LPS and KLH. RSV supplementation caused a significantincrease in the percentage of T helper cells (CD4+) in spleen from aged rats.GSH plays multiple physiological functions, including maintenance of cellularreduction-oxidation (redox) state, lipid peroxides and non-enzymatic scavenging offree radicals. GSH is synthesized de novo in a two-step process catalyzed by glutamate cysteineligase (GCL, EC6.3.2.2), formerly known as gamma-glutamylcysteine synthase (GCS), and GSHsynthase (GS, EC6.3.2.3). GCL catalyzes the first and rate-limiting step, in which glutamate isligated with cysteine to form-glutaamylcystein (-GC), which is rapidly linked to glycine to formGSH via action of GS. Semi-quantative real time-PCR (qRT-PCR) was used to assay themRNA expression of GCLc and GCLm subunit. GCL activity was determined by afluorescence assay. Protein contents were determined by Western blotting. Theseresults indicated that the decrease in GCL activity in aged rats could be reversed byRSV. Oral administration of RSV induced a remarkable increase in mRNA levels andprotein contents in livers, hearts and brains.In summary, this study highlights the anti-aging and immunostimulatoryproperties of RSV via enhancing in vivo humoral and cell-mediated immuneresponses in aged rats. It also suggests that the anti-oxidant properties of RSV viaenhancing GCL activity in vivo in aged rats. It also boosts the notion that late onset nutritional interventions with RSV may help aged organisms to generate an anti-agingeffect and improved redox state.更多还原