实验性氟中毒中枢神经系统炎症损伤机制中NF-κB信号通路改变的作用

发布时间：2018-05-28 14:00

本文选题：慢性氟中毒 + 大鼠　；参考：《贵阳医学院》2015年硕士论文

【摘要】：目的:观察慢性氟中毒动物脑组织及体外培养细胞模型中主要炎症因子肿瘤坏死因子a(tumor necrosis factora,TNFa)和白细胞介素-1b(interleukin-1b,IL-1b)的表达改变及细胞核因子-κB(nuclear factorκB,NF-κB)信号转导通路中NF-κB、NF-κB抑制蛋白α(inhibitor of nuclear factorκBα,IκBα)的表达,探讨其在慢性氟中毒炎性脑损伤机制中的作用。方法:建立慢性氟中毒动物模型:SD大鼠60只,雌雄各半,随机分为正常对照组、染氟组(饮水氟含量50 mg/L,加入氟化钠)2组;实验期为10个月;氟中毒细胞模型:体外培养THP-1细胞,分为3组,即正常对照组、低剂量染氟组(500mmol/L Na F)、高剂量染氟组(5000mmol/L Na F)。观察大鼠氟斑牙情况;用氟离子选择电极法测定大鼠尿氟及骨氟含量;用Morris水迷宫方法测定大鼠学习记忆能力;光镜下观察大鼠脑组织形态学变化(HE及尼氏染色);免疫组织化学方法检测CD11b、胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达;酶联免疫(Elisa)法检测TNFa和IL-1b含量;用蛋白印迹(Western Blotting)方法检测NF-κB p65、IκBα、Phospho-NF-κB p65和Phospho-IκB-α等因子的蛋白表达水平;用CCK8法进行细胞毒性实验,用流式细胞术检测染氟后THP-1细胞凋亡情况。对所获结果作相关统计分析。结果:1.动物实验:染氟组大鼠出现不同程度的氟斑牙,尿氟和骨氟含量明显升高;染氟组大鼠第五天逃避潜伏期较对照组显著延长,空间探索总次数显著减少,差异均具有统计学意义(P0.05);组织形态学检查显示,各组大鼠脑组织HE染色未见明显改变,但尼氏染色见染氟组大鼠脑组织神经细胞中尼氏小体染色变浅,数量变少;免疫组化结果显示,染氟组大鼠脑组织中CD11b及GFAP阳性表达较对照组明显升高,差异均有统计学意义(P0.05);染氟组大鼠脑组织中TNFa和IL-1b含量明显高于对照组(P0.05);染氟组大鼠脑组织中NF-κB p65及IκBα总蛋白表达均低于对照组(P0.05),但Phospho-NF-κB p65和Phospho-IκB-α蛋白表达均高于对照组;相关分析结果显示,染氟组大鼠脑组织中Phospho-NF-κB p65表达水平与TNFa、IL-1b含量成正比,与IκBα表达成反比。2.体外培养细胞实验:CCK8细胞毒性结果显示,用500及5000mmol/L Na F培养细胞24、48及72小时后,500mmol/L Na F及以上浓度可明显降低THP-1细胞存活率,呈现存活率与时间和剂量的负相关关系;细胞凋亡率随着染氟时间的延长逐渐增高;低、高剂量染氟组THP-1细胞炎症因子TNFa和IL-1b含量明显高于对照组(P0.05);NF-κB p65、IκBα总蛋白表达均明显低于对照组(P0.05),而Phospho-NF-κB p65和Phospho-IκB-α蛋白表达均明显高于对照组(P0.05);相关分析结果显示,低、高剂量染氟组THP-1细胞中Phospho-NF-κB p65表达水平与TNFa、IL-1b含量成正比,与IκBα表达成反比。结论:慢性氟中毒大鼠脑组织及过量氟暴露的体外培养细胞可产生大量的炎性因子及NF-κB信号通路激活,可能是过量的氟化钠蓄积导致中枢神经系统损伤的机制之一。
[Abstract]:Objective: to observe the changes of the expression of major inflammatory factors a(tumor necrosis factora1 (TNFA) and interleukin-1bsil interleukin-1b (IL-1b) in brain tissues and cultured cells of chronic fluorosis animals and to observe the inhibition of NF- 魏 B NF- 魏 B in the signal transduction pathway of nuclear factor- 魏 B(nuclear factor 魏 B and NF- 魏 B in vitro. Methods: the expression of tumor necrosis factor (TNFA) and interleukin-1b (IL-1b) in the brain of rats with chronic fluorosis were observed. Expression of protein 伪 inhibitor of nuclear factor 魏 B 伪 I 魏 B 伪 To explore its role in chronic fluorosis inflammatory brain injury mechanism. Methods: sixty male and female SD rats with chronic fluorosis were randomly divided into two groups: the control group (50 mg 路L ~ (-1) of fluoride in drinking water, added with sodium fluoride group 2), the experimental period was 10 months. Model of fluorosis cells: THP-1 cells were cultured in vitro and divided into three groups: normal control group, low dose fluoride group (500 mmol / L NaFN) and high dose fluoride group (5 000 mmol / L NAF). The fluoride content in urine and bone was determined by fluorine ion selective electrode method, and the learning and memory ability of rats was measured by Morris water maze method. The expression of CD11b, glial fibrillary acidic protein (GFAP), TNFa and IL-1b were detected by immunohistochemical method and enzyme-linked immunosorbent assay (Elisa), respectively. Western blotting method was used to detect the protein expression of factors such as Phospho-NF- 魏 B p65 and Phospho-I 魏 B- 伪, and the apoptosis of THP-1 cells after fluorosis was detected by flow cytometry. The results obtained are statistically analyzed. The result is 1: 1. Animal experiments showed that fluoride spot teeth appeared in different degrees in fluoride group, urine fluoride and bone fluorine content increased significantly, the escape latency of fluorine-exposed group on the fifth day was significantly longer than that of control group, and the total number of space exploration was significantly decreased. Histomorphological examination showed that the HE staining of brain tissue of rats in each group had no obvious change, but the staining of Nissl corpuscles in nerve cells of rats exposed to fluoride was lighter and fewer. The results of immunohistochemistry showed that the positive expression of CD11b and GFAP in the brain of rats exposed to fluoride was significantly higher than that of the control group. The contents of TNFa and IL-1b in the brain of rats exposed to fluoride were significantly higher than those of the control group (P 0.05), and the expressions of total protein of NF- 魏 B p65 and I 魏 B 伪 in the brain tissues of rats exposed to fluoride were lower than those of the control group (P 0.05), but the expression of Phospho-NF- 魏 B p65 and Phospho-I 魏 B- 伪 protein were higher than those of the control group. The results of correlation analysis showed that the expression of Phospho-NF- 魏 B p65 was directly proportional to the content of TNFa- 魏 B p65 and inversely proportional to the expression of I 魏 B 伪 in the brain of rats exposed to fluoride. The results of cell toxicity test in vitro showed that the concentration of 500 mmol / L NAF and more than 500 mmol / L NAF significantly decreased the survival rate of THP-1 cells, which showed a negative correlation with time and dose. The apoptosis rate increased gradually with the prolongation of fluorine exposure time, and decreased with the increase of fluoride exposure time. The levels of TNFa and IL-1b in THP-1 cells in high dose fluoride group were significantly higher than those in control group (P 0.05). The expression of total protein of I 魏 B 伪 was significantly lower than that of control group (P 0.05), but the expression of p65 and Phospho-I 魏 B- 伪 of Phospho-NF- 魏 B p65 and Phospho-I 魏 B- 伪 were significantly higher than that of control group (P 0.05), the correlation analysis showed that the expression of Phospho-NF- 魏 B p65 and Phospho-I 魏 B- 伪 was lower than that of control group. The expression level of Phospho-NF- 魏 B p65 in THP-1 cells exposed to high dose fluoride was directly proportional to the content of TNFa- 魏 B p65 and inversely proportional to the expression of I 魏 B 伪. Conclusion: brain tissue of rats with chronic fluorosis and cultured cells exposed to excessive fluoride can produce a large number of inflammatory factors and activation of NF- 魏 B signaling pathway, which may be one of the mechanisms of excessive accumulation of sodium fluoride leading to central nervous system damage.
【学位授予单位】：贵阳医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R595

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