【作者】 胡崇伟；

【导师】 李艳飞；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2011， 博士

【摘要】 在体实验以Wistar大鼠为实验动物,将60只体重为110g～120g的大鼠随机均分为4组,饮水染铝,三氯化铝（AlCl₃）水溶液的浓度分别为0（对照组,C组）、64.18mg/kg（低剂量组,L组）、128.36mg/kg（中剂量组,M组）、256.72mg/kg（高剂量组,H组）,连续染铝120d,复制亚慢性铝中毒大鼠模型,通过对染铝大鼠脾脏淋巴细胞增殖、免疫细胞因子、T淋巴细胞亚群、血清及下丘脑中去甲肾上腺素（NE）水平,血清中促肾上腺皮质激素释放激素（CRH）、促肾上腺皮质激素（ACTH）和皮质酮（Cort）含量,脾脏淋巴细胞内cAMP含量,脾脏淋巴细胞膜上β₂-AR密度及β₂-AR mRNA表达水平的观测,探讨染铝对大鼠免疫状态和NE水平的影响,找出NE水平与染铝大鼠免疫状态的关系;体外实验以培养1月龄的大鼠脾脏淋巴细胞为研究对象,在培养液中分别添加0（对照组,W-C组）、1/20 IC₅₀（低剂量组,W-L组）、1/10 IC₅₀（中剂量组,W-M组）、1/5 IC₅₀（高剂量组,W-H组）的AlCl₃,通过对脾脏淋巴细胞增殖、免疫细胞因子、T淋巴细胞亚群的检测,确定1/10 IC₅₀铝浓度可致淋巴细胞免疫抑制。以添加此染铝浓度的淋巴细胞为研究对象,分别添加0（对照组,N-C组）、10^-10（低剂量组,N-L组）、10^-9（中剂量组,N-M组）、10-8（高剂量组,N-H组）mol/L的NE,通过对大鼠脾脏淋巴细胞增殖、免疫细胞因子、T淋巴细胞亚群,脾脏淋巴细胞内cAMP含量,脾脏淋巴细胞膜上β₂-AR密度及β₂-AR mRNA表达水平的检测,探讨NE对铝免疫毒性的调节作用。实验结果如下:体内实验结果表明:1.亚慢性铝中毒大鼠血清和脾脏中铝含量均显著或极显著高于C组;T、B淋巴细胞增殖率,CD3⁺、CD4⁺、CD8⁺ T淋巴细胞表达率和CD4⁺/CD8⁺的比值以及脾脏组织中IL-2和TNF-α含量均显著或极显著低于C组,说明铝可致大鼠免疫功能抑制。2.亚慢性铝中毒大鼠血清中NE含量、脾脏淋巴细胞膜上的β₂-AR密度及β₂-AR mRNA表达水平和淋巴细胞内cAMP含量均显著或极显著高于C组,说明铝能增加外周NE水平,并激活β₂-AR-cAMP途径,直接抑制大鼠免疫功能。3.亚慢性铝中毒大鼠下丘脑中NE含量显著低于C组,血清中CRH、ACTH和Cort含量显著或极显著高于C组,说明铝能抑制突触前膜对NE的再摄入,提高细胞外NE水平,进而通过激活下丘脑-垂体-肾上腺（HPA）轴,间接抑制免疫功能。4.亚慢性铝中毒大鼠血清中NE水平与T、B淋巴细胞增值率,脾脏组织中IL-2和TNF-α含量,CD3⁺、CD4⁺T淋巴细胞表达率以及CD4⁺/CD8⁺的比值均呈显著负相关,说明NE与铝的免疫毒性密切相关,外周高水平NE可加重铝引发的免疫抑制。体外实验结果表明:1.应用MTT比色法测得染铝24h后大鼠脾脏淋巴细胞的半数抑制浓度(IC₅₀)为5.52mmol/L,95%可信区间为4.76mmol/L～6.38mmol/L。2.随染铝剂量的增加,大鼠T、B淋巴细胞增殖率,CD3⁺、CD4⁺、CD8⁺ T淋巴细胞表达率和CD4⁺/CD8⁺的比值以及淋巴细胞分泌IL-2和TNF-α含量均逐渐降低,W-M组、W-H组显著或极显著低于W-C组,W-L组与W-C组比较差异不显著,说明铝能抑制淋巴细胞功能,且因为W-M组能引起显著的免疫抑制,故选择W-M组作为后续实验的研究对象。3.随染铝剂量的增加,大鼠脾脏淋巴细胞膜上的β₂-AR密度及β₂-AR mRNA表达水平和淋巴细胞内cAMP含量高于W-C组,且W-M组、W-H组均极显著高于W-C组,同时伴随淋巴细胞免疫功能抑制,说明β₂-AR-cAMP途径是铝免疫毒性的机制之一。4.随NE浓度的增加,淋巴细胞细胞内cAMP含量显著高于N-C组,大鼠T淋巴细胞增殖率,CD3⁺、CD4⁺、CD8⁺ T淋巴细胞表达率和CD4⁺/CD8⁺的比值以及淋巴细胞分泌IL-2和TNF-α含量均显著或显著低于N-C组,B淋巴细胞增值率与N-C组比较无显著变化,说明NE可通过β₂-AR-cAMP途径加重铝对T淋巴细胞的抑制作用,但对B淋巴细胞没有显著影响。更多还原

【Abstract】 In vivo experiment, Wistar rats were used as experimental animals. 60 healthy rats （110g～120g） were randomly divided into 4 groups, and were orally exposed to 0 （control group, C group）, 64.18 （low dose group, L group）, 128.36 （middle dose group, M group） and 256.72 （high dose group, H group） mg/kg body weight aluminum trichloride （AlCl₃） in drinking water for 120 days, respectively. And sub-chronic Al intoxication model was built successfully. The spleen lymphocyte proliferation, immune cytokines, T lymphocyte subsets, NE levels in serum and hypothalamus, corticotropin release hormone （CRH）, adrenal cortex hormone （ACTH） and cortisone （Cort） content in serum, the cAMP content in spleen lymphocyte, the density ofβ₂-AR on spleen lymphocyte membrane and expression level ofβ₂-AR mRNA were detected to study the influence of aluminum exposure on immune state and NE level of rats and to find the relationship between NE level and the immune state of aluminum exposed rats.In vitro experiment, the spleen lymphocyte of 1-month-old rats was obtained. The culture solutions were added with AlCl₃ of 0 （control group, W-C group）, 1/20 IC₅₀ （low dose group, W-L group）, 1/10 IC₅₀ （middle dose group, W-M group） and 1/5 IC₅₀ （high dose group, W-H group）, respectively. The detection of in vitro proliferation of rats’spleen lymphocyte, immune cytokines and T lymphocyte subsets were detected, and the results showed that aluminum with concentration of 1/10 IC₅₀ could inhibit lymphocyte immune function in vitro. The lymphocyte with this concentration was chosen and added with NE of 0 （control group, N-C group）, 10-10 （low dose group, N-L group）, 10-9 （middle dose group, N-M group） and 10-8 （high dose group, N-H group） mol/L, respectively. And the spleen lymphocyte proliferation, immune cytokines, T lymphocyte subsets, the cAMP content in spleen lymphocyte, the density ofβ₂-AR on spleen lymphocyte membrane and expression level ofβ₂-AR mRNA were detected to study the regulation function of NE on aluminum immunotoxicity. The experiment results showed as follows:The result of the vivo experiment indicated that:1. The aluminum contents in serum, spleen and brain tissue of sub-chronic aluminum poisoned rats were all extremely significantly higher than C group; the expression rate of CD3⁺, CD4⁺, CD8⁺ T lymphocyte, proportionality of CD4⁺/CD8⁺, IL-2 and TNF-αcontents from lymphocyte secretion were all significantly lower than C group, which showed that aluminum could inhibit the immune function of rats. 2. The NE content in serum, the density ofβ₂-AR on spleen lymphocyte membrane, expression level ofβ₂-AR mRNA and cAMP content in lymphocyte of sub-chronic aluminum poisoned rats were all significantly higher than C group, which showed that aluminum could increase the peripheral NE level, activate the approach ofβ₂-AR-cAMP, and inhibit immune function directly.3. The NE content in the brain tissues of sub-chronic aluminum poisoned rats was significantly lower than C group, while the CRH, ACTH and Cort contents in serum were all significantly higher than C group, which showed that aluminum could inhibit the NE re-englobement of presynaptic membrane, raise the extracellular NE level, and inhibit immune function indirectly by activating the HPA axle.4. The correlation analysis results between NE level in the spleen of sub-chronic aluminum exposed rats and proliferation rate of T/B lymphocyte, IL-2 and TNF-αcontent in spleen, the expression rate of CD3⁺, CD4⁺ T lymphocyte and proportionality of CD4⁺/CD8⁺ were all significantly negatively correlated, which showed that NE was closely related to aluminum immunotoxicity, and extracellular high-level NE could aggravate immunosuppression induced by aluminum.The result of the vitro experiment indicated that:1. Exposed to aluminum for 24 h in vitro, the IC₅₀ of AlCl₃ to rat splenic lymphocytes detected by MTT was 5.52 mmol/L, and 95% confidence interval was 4.76 mmol/L～6.38 mmol/L.2. With the increasing of the aluminum, the proliferation rate of T/B lymphocyte of rats, the expression rate of CD3⁺, CD4⁺, CD8⁺ T lymphocyte, proportionality of CD4⁺/CD8⁺, IL-2 and TNF-αcontent from lymphocyte secretion were all gradually decreased. And the W-M and W-H groups were significantly lower than C group, while the difference between W-L group and with W-C group was not significant. The results proved further that aluminum could inhibit the function of lymphocyte. Because W-M group could cause remarkable immunosuppression, W-M group was chosen as the research object for future experiment.3. With the increasing of aluminum, the density ofβ₂-AR and expression level ofβ₂-AR mRNA on lymphocyte of rats were higher than that in W-C group and the W-M and W-H groups were significantly higher than that in W-C group. And the inhibition of lymphocyte immune function was accompanied. The results showed that theβ₂-AR approach was one of the aluminum immunotoxicity mechanisms.4. With the increasing of NE, the cAMP content in lymphocyte was significantly higher than that in N-C group, and the proliferation rate of T lymphocyte of rats, the expression rate of CD3⁺, CD4⁺, CD8⁺ T lymphocyte, proportionality of CD4⁺/CD8⁺, IL-2 and TNF-αcontents from lymphocyte secretion were all significantly lower than that in N-C group. B lymphocyte proliferation rate had no significant change compared with N-C group. The results showed that theβ₂-AR-cAMP approach could be used to aggravate the inhibitory effect of aluminum on T lymphocyte, but it would have no remarkable influence on B lymphocyte.更多还原