【作者】 毕明刚；

【导师】 张永祥； 周文霞；

【作者基本信息】 中国人民解放军军事医学科学院 ， 药理学， 2004， 博士

【摘要】 自医学产生以来,对衰老本质的探索和抗衰老药物的寻找始终是医药学家的不懈追求。衰老是全身多器官、多系统功能衰退的综合表现,其中免疫系统最早出现组织结构改变和功能紊乱,并通过神经内分泌免疫调节(Neuroendocrine-immuuno-modulation, NIM)网络与神经、内分泌等其它系统相互作用,对机体整体机能产生影响。近年来研究表明,细胞Notch信号转导通路对T细胞的分化进程具有重要影响,同时与免疫衰老密切相关。在Notch通路上存在着多种信号分子,其中以Notch受体、早老蛋白(Presenilin, PS)、人分裂加速因子(Human enhancer of split, HES1)的作用较为重要,它们表达水平的改变可直接影响并反映Notch信号的激活程度。但是目前Notch信号通路中各种信号分子的变化与免疫衰老过程中细胞免疫反应、T细胞分化之间的相互关系尚未得到阐明。 快速老化模型小鼠(Senescence accelerated mouse, SAM)是一种出生后全身各系统功能迅速老化的模型小鼠。SAMP8(SAM-prone/8)是SAMP系(快速老化)的一个亚系,其特征是随增龄而出现快速、进行性中枢学习和记忆功能和免疫功能衰退,SAM-resistance/1(SAMR1),抗快速老化亚系,为SAMP的正常对照亚系。我室大量研究表明,中医滋补肾阴的经典方剂——六味地黄汤(Liuwei Dihuang decoction, LW)对多种原因所致免疫功能低下均具有明显改善作用,口服给予LW对SAMP8的免疫功能和中枢学习记忆功能均具有明显改善作用,对其内分泌平衡失调等亦具有改善作用,表明LW具有延缓衰老的作用。 我室多年来一直从事NIM的研究,探讨了衰老过程中早老蛋白、β淀粉样蛋白前体蛋白等基因与某些神经退行性疾病如阿尔茨海默病的关系。在以上基础上,本研究重点观察了SAMP8增龄过程中胸腺淋巴细胞免疫功能的动态变化过程,进而研究了Notch信号转导通路信号分子基因表达与胸腺淋巴细胞分化的关系,并初步观察了LW及其拆更多还原

【Abstract】 It is the continuous pursuit for exploring the essence of senescence and anti-senescence drugs since the medicine began. Senescence is the comprehensive manifestation of multiple organic and systematic hypofunction, during which disorders of tissue structure and functic", of immunity system occur earliest and organic function is influenced through neuroendocrine -immuno-modulation network.Recent studies have revealed the close relationship between Notch signal transduction pathway and immunosenescence and significant influences of the pathway on T cell differentiation. Changes of expression of signaling molecules on Notch pathway, such as Notch receptor, presenilin (PS) and Human enhancer of split (HES1), can influence and indicate degree of Notch signaling activation. But correlation of cell immune responses, T cell differentiation during immunosenescence and changes of signaling molecules in Notch pathway is still unclear.Senescence accelerated mouse (SAM) is manifested as accelerating senescence after birth and is cultured being for presenting premature aging in process of inbred strain. SAMP8 (Senescence accelerated mouse/prone8) is a kind of substrains of SAMP, which is characterized by learning and memory impairment corresponding to age-related change, as well as immunosuppression. SAMRl (Senescence accelerated mouse/resistance 1), a kind of substrain of anti-accelerated-senescence mouse, is the contrast substrain to SAMP.Liuwei Dihuang decoction (LW), the classic formula of tonifying kidney yin, can improve immunologic hypofunction obviously. Oral intake of LW is proved to have obvious anti-senescence action in SAMP8 by improving immunologic function, learning and memory ability, and endocrine disequilibrium.Our group has been engaged in studying NIM for long time and has explored therelationship between presenilin, β-Amyloid precursor protein gene, etc., and certain nervous degeneration diseases, such as Alzheimer’s disease (AD).Based on above-mentioned studies, age-related dynamic changes in immunologic function of thymocytes in SAMP8 were observed and the relationship between gene expression correlated to Notch signal molecules and thymocyte differentiation was explored. Furthermore, influences of LW and its decomposed recipes on thymocytes subsets and gene expression related to Notch signaling molecules in normal Balb/c mouse and SAM were primarily observed. The purpose is to illuminate the relationship between immunosenescence and Notch signaling transduction pathway in the process of aging, so as to provide experimental basis for recognizing the mechanism and process of immunosenescence.The study included four parts. They are changes of thymus structure and T cell function correlated to SAMP8 aging, dynamic changes of thymocyte differentiation in SAMPS age-increasing, dynamic changes of gene expression of signaling molecules related to Notch pathway and influences of LW and its decomposed recipes (SB and SX) on gene expression of relative signaling molecules and thymocyte subsets in normal mouse and SAMP8.Ⅰ Changes of thymus atrophy and T cell function during aging of SAMThymus is an important organ in the immune system in which T cells development takes place. Obvious changes of thymic structure and function, especially age-related atrophies, take place in the process of aging. Thymic atrophy undoubtedly influences thymic T cell differentiation and subsequently influences quantity and function of mature T cell from thymus to peripheral lymphoid organs. Age-related changes of thymic tissue structure and thymocyte proliferation were observed in SAM in the study.The results show that thymuses of SAMR1 and SAMP8 come to enlarge after birth and peaks at 4W. After that, age-related atrophy occurs and becomes much obvious at 8W. Before 4W, weight and tissue structure of thymus in SAMP8 is similar to those in SAMR1. At 6W, thymus weight of both SAMR1 and SAMP8 decrease in some extent, which is more obvious in SAMP8 than that in SAMR1. Thinner cortex and decreased cell amount can be found in tissue slices of SAMP8. At 8W, thymic weight, thinner cortex and cell amount decreased further in SAMP8. The results indicate that speed and extent of thymus atrophy in SAMP8 is much more severe than that in SAMR1.Changes of SAM thymocyte proliferation in age-related process were further observed. The results show that thymocyte proliferation increases in SAMR1 and SAMP8 at 6W. There is no significant difference between SAMP8 and SAMR1 at 1 and 2week. While at 4W, thymocyte proliferation in SAMP8 is lower obviously than that in SAMR1, indicating that thymic function starts to lower.Ⅱ Dynamic changes of thymocyte differentiation during aging of SAMIn the process of aging, thymocytes in mice develop from CD4-CD8- double negative (DN) to CD4+CD8+ double positive (DP), then to CD4+ single positive (SP) or CD8+ single positive (SP). CD4+ single positive (SP) is helper T lymphocyte (Th) and CD8+ single positive (SP) is suppressor T lymphocyte (Ts) and cytotoxic T lymphocyte (Tc). T cell subset with helper function in CDg+ cells has been found in recent studies. Furthermore, CD28 is an accepted important symbol of immunosenescence. In the study, flow cytometry (FCM) is used to observe changes of thymocyte subsets in the process of aging in SAM.The results show that ratio of CD4+/CD8+ cells reduces gradually in the process of aging in SAMR1 and SAMP8 thymus. CD4+/CD8+ cells reduce obviously in mice from 6W to 8W without obvious difference. Ratio of CD4+/CDg+ SP cells increases age-relatively. Ratios of CD4+ cells in SAMP8 and SAMR1 thymus at lW and 2W are low, increase gradually at 4W and 6W and obviously at 6W and 8W without obvious difference. Ratio of CD8+ cells increases gradually and there is no obvious difference in SAMP8 and SAMR1 thymus at lW and 2W. In mouse from 4W to 8W, the ratio in SAMP8 is much higher than that in SAMR1 and the difference becomes larger and larger age-relatively.As to the ratio of CD4+/CD8+, in SAMR1, it is lower at 1W, increase at 2W, changes insignificantly at 2W, 4W, 6W, and increases with great extent at 8W. In SAMP8, the ratio does not change notably at lW, 2W, 4W and 6W, while it greatly upregulates at 8W. There is no obvious difference between the two groups at 1W, since the second week the ratio is lower in SAMP8 than that in SAMR1, and the difference maintains to 8W, particularly at 8W.Age-related changes of CD28 expression of SAM thymocyte are observed. Tendencies of CD28 fluorescence index of SAMR1 and SAMP8 thymocytes are similar. The index greatly increases at 1W and 2W, slightly increases at 4W and 6W, and then increases obviously from 6 to 8W. Compared with age-matched SAMR1, it is obviously higher in SAMR8 than that in SAMR1 at every stage.The results show that the processes of thymocyte proliferation in SAMR1 and SAMP8 are similar. Compared with age-matched SAMR1, there is no obvious difference of changes in ratio of CD4+thymocytes in SAMP8, but ratio of CD8+ cell in SAMR1 at 4W is obviously lower than that in age-matched SAMR1, causing reduction of CD4+/CD8+ ratio. It indicates that immunologic disequilibrium occurs in SAMP8 at 4W. Furthermore, CD28 expression of thymocytes in SAMP8 from 1W to 8W is much higher than that in age-matched SAMR1, indicating higher degree of thymocyte senescence in SAMP8 than in SAMR1. That is the one of the possible causes for hypofunction and differentiation disequilibrium of thymocyte subsets.Ⅲ Dynamic changes in gene expression of relative signaling molecules on Notch pathway in process of aging of SAMAt present, the mechanism of immunosenescence of SAMP8 is still unclear. No report has been published about the relationship between thymocyte subsets and Notch signal transduction during immunosenescence in SAMP8. This study, which is tested in SAMP8 and SAMR1 at different week age (1W, 2W, 4W, 6W, 8W), detects the thymocyte subsets and gene Notchl, PS1, PS2 and HESl mRNA expression by techniques of FCM and Real-time quantitative RT-PCR, meanwhile, observes the dynamical changes of thymocytes subsets and gene expression of Notchl, PS1, PS2 and HES1 in Notch signal transduction pathway in SAMP8 and SAMR1 thymus, and analyzes the correlation between them as well.i Dynamic changes in gene Notchl expression of thymocytesThe results show that Notchl gene expression in SAMR1 and SAMP8 thymocytes presents the similar trend of age-related increasing. Notchl expression is significantly higher in SAMP8 than that in age-matched SAMP1 at every stage.ii Dynamic changes in gene PS1 expression of thymocytesIt is found that expression of gene PS1 in SAMR1 and SAMP8 thymocytes shows gradual decreasing tendency in age-related process. Since the second week, PS 1 gene expression of SAMP8 thymocytes drops abruptly and is notably lower than that in age-matched SAMR1.iii Dynamic changes in gene PS2 Expression of thymocytesPS2 gene expression in SAMR1 and SAMP8 thymocytes shows an age-related increasing tendency. After 4W, PS2 gene expression in SAMP8 thymocytes is obviously higher than that in SAMR1.iv Dynamic changes in gene expression of Hes1 of thymocytesGene expression of Hes1 in SAMR1 and SAMP8 thymocytes shows the age-related increasing tendency. At 1W and 2W, the expression of HES1 in SAMP8 is similar with that in SAMR1. After 4W, Hesl gene expression in SAMP8 thymocytes is obviously higher than that in age-matched SAMR1.v Analysis of correlation between thymus CD8+ subsets and gene expression of Notchl, PSl, PS2 and HESl during aging of SAMThe correlation between thymus CD8+ subsets and gene expression of Notch1, PS1, PS2 and HES1 is analyzed with Pearson relative factor method. It shows that, the relationship between SAMR1 CD8+ thymocyte subsets percentage and expression of Notchl, PSl, PS2 and HES1 are respectively Rnotchi=0.73 (P=0.002), RPS1=-0.70 (P=0.004), RpS2=0.68 (p=0.006), Rhes1=0.68 (P=0.006), and the relationship between SAMP8 CD8+ thymocyte subsets percentage and Notch1, PS1, PS2 and HES1 expression respectively are RnotchI=0.77(P=0.001), Rps1=-0.77(P=0.001), RPS2=0.70(P=0.005), RHEs1=0.73(P=0.002).The above results indicate changes of gene expression of Notch signaling molecules during aging in SAM. Expression of Notchl, PS2 and HES1 increases age-relatively and expression decreases. Analyses of correlation between CD8+ ratio and molecular gene expression show that CD8+ thymocyte differentiation is positively correlated with expression of Notch1, PS2 and HES1 and negatively correlated with expression of PS1, revealing the close relations between thymocyte subsets differentiation and gene expression of Notch signaling molecules. It is also found that gene expression of Notchl in SAMP8 thymocytes is much higher than that in age-matched SAMR1 and expression of PS2 and HES1 is much higher than that in age-matched SAMR1 after 4W, indicating that abnormal gene expression of Notch signaling molecules is one of the importantly possible causes for hypofunction and differentiation disequilibrium of SAMP8 thymocyte subsets.Ⅳ Effects of LW and its decomposed recipes on thymocyte subsets and gene expression of relative signaling molecules on Notch pathway in micei Effects of LW and its decomposed recipes on thymocyte subsets and gene expression of relative signaling molecules on Notch pathway in normal micea. Effects on thymocyte subsets and CD28 expression in normal miceChanges of thymocyte subsets are observed after continuous oral intake of LW (5, 10 and 20g/kg), SB(6.4g/kg) and SX(3.6g/kg) for 2 weeks in mice. The results show that ratios of CD4+CD8+ thymocytes and CD4+ thymocytes are slightly influenced by LW, SB and SX, while LW of three dosages can not only increase ratio of CD8+ thymocytes, but decrease CD4+/CD8+ratio obviously. Action of LW of dosage 10g/kg is the most obvious. Ratio of CDg+ thymocytes is slightly influenced by SB and SX. Gene expression of CD28 thymocyte is increased by LW of low and middle dosage, SB and SX. Action of LW of low dosage is most obvious and influence of LW of high dosage is slight.b. Effects of LW and its decomposed recipes on gene expression of relative signaling molecules on Notch pathway of thymocytes in normal Balb/c miceDosages and feeding method of LW, SB and SX are the same as above section. The results indicate that gene expression of Notchl and PS1 of Balb/C mice thymocytes is not influenced by LW (5,10 and 20g/kg), SB (6.4g/kg) and SX (3.6g/kg). Comparatively, LW of three dosages can increase gene expression of PS2 and HES1 obviously and dose-effect relatively. However,, there is no obvious effect of decomposed recipe of LW, namely SB and SX on PS2 and HES1 gene expression.ii Effects of LW and its decomposed recipes on thymocyte subsets and gene expression of relative signaling molecules on Notch pathway in SAMP8a. Effects on thymocyte subsets and CD28 expression in SAMP8Influences on gene expression of Notch signaling molecules and thymocyte subsets in SAMP8 at 4W by oral intake of LW, SB and SX are observed in the experiment. There shows no obvious effect on SAMP8 thymus CD4+CD8+ DP cell after continuous four-week oral taking of LW (10g/kg) , SB (6.4g/kg) and SX (3.6 g/kg). LW can increase ratio of CD4+ thymocytes in SAMP8 without statistic difference. SB and SX can not influence CD4+ ratio. CD8+ thymocyte ratio in SAMP8 can be decreased remarkably after taking LW and SB and CD4+/CD8+ ratio is increased. But after the oral taking of SX, there is no significant effect on CD8+ thymocyte ratio in SAMP8. LW, SB and SX can increase CD28 expression obviously. Among which, action of SB is the best.b. Effects of LW and its decomposed recipes on gene expression of relative signaling molecules on Notch pathway of thymocytes in SAMP8After continuous four-week oral taking of LW (10g/kg), SB (6.4g/kg) and SX (3.6g/kg), different effects on four Notch signaling molecules of SAMP8 thymocytes are observed. There are no obvious changes on gene expression of Notch and PSl in SAMP8 thymocytes by LW, SB and SX. As for the gene expression of PS2 and HES1, it can be reduced to different extent. Action of LW is the strongest.The results show that LW can obviously increase ratio of CD8+ thymocytes and decrease CD4+/CD8+ ratio in Balb/c mice But LW and SB can obviously decrease ratio of CD8+ thymocytes and increase CD4+/CD8+ ratio in SAMP8. It indicates that LW can act on differentiation of thymocyte subsets in Balb/c miceand SAMP8 differently. On the effect of the gene expression of Notch signaling molecules of thymocytes, LW can obviously increase the gene expression of PS2 and HES1 in Balb/c mice, but the LW SB and SX can obviously decrease the gene expression of PS2 and HES1 in SAMP8.It shows It is different effect of LW and its decomposed recipes SB, SX on gene expression related to Notch signals of thymocytes in Balb/c mice and SAMP8, but it is same effect of LW and its decomposed recipes SB, SX on thymocytes CD8+ cell in Balb/c mice and SAMP8.In a word, there has immunosenescence in SAMP8 in the process of thymus atrophy corresponding to age-related changes. The differentiation of thymocyte subsets is tendency to CD8+ SP cell, the immunity function decreased, compared with SAMR1, SAMP8 is earlier in the immunosenescence state. The change is related to the gene expression of Notch signal transduction pathway. As well that it is positively related to gene expression of Notchl, PS2 and HES 1, and negatively related to PS 1.It is part of similar and part of different for the effect of LW and its decomposed recipes on thymocytes subsets in Balb/c mice and SAMP8, as well as on gene expression related toNotch signals. For example, both of LW, three tonifying ingredients (SB) and three reducing ingredients (SX) can promote the expression of thymocytes CD28 in two different kinds of mice. The difference lies in that LW can stimulate the differentiation of thymus CD8+ SP cell in Balb/c mice, and increase the expression of gene PS2 and HES1. Whereas, LW and three tonifying ingredients(SB)inhibit the premature differentiation of thymus CD8+ SP cell, downregulate the expression of gene PS2 and HES1. The results indicate LW owns two-way adjustment. The combination of three tonifying (SB) and three reducing (SX) can restrict mutually and produce new biological effects, which embodies the scientific compatibility.The above-mentioned suggest that, immunosenescence is closely correlated to CD8+ SP cell ratio, and the premature differentiation of CD8+ SP is related to Notch signals as well. Regulating and controlling the gene expression relative to Notch signals can inhibit the premature differentiation of CD8+ SP cell, so as to delay the immunosenescence.更多还原