【作者】 钱露；

【导师】 赵茹茜； 陈杰；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2003， 硕士

【摘要】 本实验观察3、7和13月龄大鼠胸腺增龄性退化的表观特征，并观察外周血T细胞功能的增龄性变化，同时通过定量检测大鼠胸腺IGF-1、IGF-1R和雌激素受体mRNA的表达，以探讨内源性IGF-1和雌激素调节胸腺增龄性退化的机制。此外，观察生理调节剂——半胱胺和大豆黄酮对卵巢切除大鼠胸腺退化和胸腺IGF-1、IGF-1R mRNA表达的影响。 一、大鼠胸腺退化的表观特征与外周血T细胞功能变化 大鼠胸腺重量和胸腺指数在3月龄后均发生极显著下降(P＜0.01，n=12)，13月龄和7月龄比较胸腺重量和胸腺指数无显著差异。胸腺组织形态结构也显示出与胸腺重量相应的时序变化模式，3月龄时大鼠胸腺分成若干小叶，皮髓界线清晰，7月龄和13月龄皮髓界限消失，分叶不明显，叶间结缔组织增多，有较多的脂肪细胞侵润，表现出明显的胸腺萎缩特征。 大鼠外周血T淋巴细胞衰老和胸腺退化进程不一。在无PHA诱导情况下，3、7和13月龄的淋巴细胞自然增殖活力无显著差异；在PHA诱导下，7月龄大鼠T淋巴细胞转化活力显著高于3月龄(P＜0.05，n=5)，13月龄较7月龄略有下降趋势，但不显著(P=0.169，n=5)。各月龄组间血清IL-2含量无显著差异，3～7月龄略有上升趋势(P=0.122，n=12)。提示T细胞衰老比胸腺退化延缓，3月龄时外周T淋巴细胞功能处于暂时性的抑制状态。 二、IGF-1和雌激素调节大鼠胸腺增龄性退化的机制 用相对定量RT-PCR方法检测，胸腺IGF-1R mRNA表达3月龄时极显著高于7和13月龄(P＜0.01，n=7)，与大鼠胸腺指数的增龄性变化模式一致，呈极显著正相关(R~2=0.727，P＜0.001)，提示IGF-1R是胸腺退化的主要调节者。胸腺IGF-1 mRNA表达呈现随月龄增长而上升的趋势，13月龄组显著高于3月龄组(P＜0.05，n=7)，与胸腺指数呈弱负相关(R~2=0.201，P=0.087)，与IGF-1 mRNA表达呈中度负相关(R~2=0.203，P=0.076)，提示胸腺存在IGF-1与IGF-1R的负反馈调节环路。 钱翼 胸腺IGFI、IGFIR和雌激素受体MA表达与胸腺退化的关系 本实验首次检测到 ER a、ER 01和 EM 2在大鼠胸腺的共同表达。在胸腺退化中，ER a InRNA表达无显著性变化；ER pl和ER p 2 InRNA表达丰度接近，3月龄时其表达丰度相对较高，7和13月龄较低，这与胸腺重量变化趋势相同；同时实验显示血清雌激素水平与胸腺指蛐显著负相关（R＝＝－0．747，P吻．of），提示血液雌激素可能通过反馈下调 ER p InRNA表达而促进胸腺退化，而 ER a在胸腺退化过程中可能起自稳态作用．三、半用和大豆黄酮对卵巢切除大鼠胸腺退化和外周血T淋巴细胞功能的影响 半月u去使卵巢切除大鼠胸腺指数显著增加（P＜0．05，n司），外周血淋巴细胞自然增殖活力显著提高（P＜0．05，n＝＝5），而对T淋巴细胞转化活力无显著影响．半瑞胶机腺 IGF－IR rnRNA表俄显著上调（P＜0．of，nd），6 I怔1 表达显著下调门＜0．05）．进一步提示购机在 IGF－l与 IGF－IR的负反馈调节环路，IGF－IR上调可能是半眈胺逆转胸腺萎缩的原因之一． 林大豆黄酮和半跳胺与单独添加半既胶比较，胸腺IGF上 mRNA表这有所下降（P＝（刀67，n＝6），但胸腺重量和胸毗数未发生变化．在添加半耽胶的树上添加 大豆黄酮对外周血淋巴细胞自然增殖活力无显著影响． 实验结果表明，在大鼠胸腺增龄性退化过程中，系统 IGF－l和雌激素通过下调IGF1 和ER p表达参与胸腺退化，胸减部IGF并对其受体有负助调节作用．半 晌能够上调胸腺 IGF1 InRNA表达逆转胸腺退化，同时促进外周 T细胞功能；在 添加半脱胶的动上添加大豆黄国胸腺RIF4 表达下调，提示半眈胶和大豆 黄酮对胸响相互桔抗作用．更多还原

【Abstract】 To gain more insights into the mechanism of endogenous IGF-1 and estrogen action on thymic involution, this study was designed to expatiate mRNA expression patterns of thymic IGF-1, IGF-IR and estrogen receptors (ERs) in 3-, 7-and 13-month-old rats. At the same tune, changes of peripheral blood T cell function with age were observed. Furthermore, effects of cysteamine and daidzein on thymic involution and mRNA expression of thymic IGF-1, IGF-IR and ERs were tested in ovariectomized rats.1. The characteristics of rat thymic involution and changes of peripheral blood T cell function with ageThe thymic weight and thymic index of 3-month-old rat were significantly higher than that of 7- and 13-month-old rats (P<0.01, n=15), but no significant difference was observed between 3- and 7-month-old rats. This age-related change was mirrored by the histological results. Thymus of 3-month-old rats showed well-developed lobules with clear borderlines between cortex and medulla, compared with 7- and 13-month-old counterparts in which severe atrophic morphological characteristics were shown with vague borderlines and increased connective tissues.The aging progress of peripheral blood lymphocyte didn’t coincide with thymic involution. The natural lymphocyte proliferation was not significantly different among 3 age groups of rats, so was serum IL-2 concentration. The lymphocyte transformation induced by PHA in 7-month-old rats was markedly higher than that in 3-month-old rats (P<0.05, n=5). All above results suggested that T cell senesced after the thymic involution, and T lymphocyte transformation at 3 months of age was inhibited temporarily.2. The mechanisms of IGF-1 and estrogen action on thymic involution in ratsRelative RT-PCR was used to detect mRNA expression in the present study. ThymicIGF-1R mRNA expression dropped sharply from 3 months of age onward (P<0.01, n=6), but didn’t differ significantly between 7 and 13 months of age, and this positively correlated with thymic index (R2=0.727, P<0.001). This result implied that thymic IGF-1R may be a major modulator during rat thymic involution. On the contrary, thymic IGF-1 mRNA expression increased with age, which was negatively correlated with thymic index (R2=0.201, P=0.087), and with thymic IGF-1R mRNA expression (R2=0.203, P=0.076), suggesting a negative feedback loop of thymic IGF-1 and IGF-1R.ER a , ER β 1 and ER P 2 mRNA were firstly found to be expressed simultaneously in rat thymus. The mRNA expression of thymic ER a was not significantly different among 3 age groups. The relative abundances of ER β 1 and ER β 2 mRNA were comparable in rat thymus, both declined with age. This coincided with the increase of serum estrogen concentration. Above results indicated that estrogen may control thymic involution by down-regulating thymic ER β expression. The thymic ER a expressed consistently during thymic involution.3.The effects of cysteamine and daidzein on ovariectomized rat thymus and peripheral blood T lymphocyteDiet supplementation of cysteamine increased thymic weight (P<0.05, n=15) and thymic IGF-1R mRNA expression (P<0.01, n=6), but decreased thymic IGF-1 mRNA expression in ovariectomized rats. It suggested that cysteamine reverse the atrophic process of thymus by up-regulating thymic IGF-1R expression. Cysteamine enhanced natural proliferation of peripheral blood lymphocytes, but not PHA-stimulated lymphocyte transformation.Diet supplementation of cysteamine plus daidzein decreased (P=0.067, n=6) IGF-IR mRNA expression of ovariectomized rats compared to cysteamine treated group, although no differences in thymic weight and thymic index were observed between two groups. It implied that daidzein influence, to some extant, the effect of cysteamine on thymic gene expression.To summarize, during rat thymic involution in aging, endogenous IGF-1 and estrogen may control this progress mainly via their receptor mechanism, i.e. down-regulating IGF-IR and ER β gene expression respectively. Thymic IGF-1 and IGF-IR may form a更多还原