高氟干扰T淋巴细胞分化定向蛋白质相互作用研究

发布时间：2019-01-15 21:22

【摘要】：地氟病作为目前我国流行最广泛,并且严重危害到人类健康与动物生命的一种的地方病和多发病,是由长时间摄入高浓度的氟引起的。大量研究表明,高氟可造成机体的广泛性损伤,包括免疫系统。本研究通过向小鼠饮水中添加不同剂量的氟(0mg/L,50mg/L,100mg/L)建立动物模型。采用透射电镜、流式细胞术、i TRAQ和Real-time PCR等技术,从血清学,组织病理形态学,细胞生物学和蛋白组学等角度研究了高氟的免疫毒性作用。具体结果如下:1.氟对小鼠血常规和胸腺超微结构的影响高氟可抑制小鼠的体增重,并造成胸腺超微结构的损伤,表现为核染色质减少、固缩,细胞膜核膜的双层膜结构不明显,细胞质浓缩出现空泡化,线粒体脊断裂、结构破坏等。血常规检查结果显示高氟可导致小鼠的外周血WBC数、淋巴细胞数、中间细胞数和粒细胞数目显著下降,改变淋巴细胞及粒细胞的比例,表明氟可以影响机体的免疫状态,这与胸腺损伤的结果相一致。2.氟对小鼠T细胞亚群的的影响采用流式细胞术检测小鼠胸腺T细胞亚群的变化,结果显示,高剂量的氟可导致胸腺CD4+T细胞和CD8+T细胞的比例下降,且差异显著,而DP细胞在两个氟处理组内的比例上升,DN细胞在氟处理组中有下降趋势,证实了高氟可以影响胸腺T细胞的分化,抑制了CD4+或CD8+单阳性细胞的生成。3.氟对小鼠胸腺蛋白质与蛋白质相互作用的影响i TRAQ实验结果显示,高氟诱导350多个蛋白发生差异表达。进一步的生物信息学分析表明,这些蛋白主要分布于110多个信号通路,其中参与细胞代谢的有30多条,涉及70多个差异蛋白;参与信号调控通路的有50多,涉及160多个差异蛋白,参与疾病发生的有19条通路,涉及60多个蛋白。这些蛋白表达的紊乱可以影响胸腺细胞的生物过程,进而干扰T细胞的分化。4.氟对小鼠T细胞分化关键基因m RNA表达的影响实时荧光定量PCR技术验证差异蛋白m RNA表达影响的结果显示,高氟可抑制Rps3、Rps3a及Pfn1m RNA的表达,并促进Nefh和Nefm的m RNA表达,与蛋白表达检测结果相一致。综上所述,高氟可以造成胸腺超微结构的损伤,并影响胸腺T细胞分化发育,引起大量胸腺内蛋白的差异表达。本试验从蛋白组学的角度出发,研究了高氟对T淋巴细胞分化的影响,为进一步阐明氟干扰T细胞分化定向的机制奠定了基础。
[Abstract]:As one of the most prevalent endemic diseases in China and seriously endangering human health and animal life, endemic fluorosis is caused by a long time intake of high concentrations of fluorine. Numerous studies have shown that high fluoride can cause widespread damage to the body, including the immune system. In this study, animal models were established by adding different doses of fluoride (0 mg / L, 50 mg / L, 100 mg / L) to the drinking water of mice. The immunotoxicity of high fluoride was studied by transmission electron microscopy (TEM), flow cytometry (FCM), i TRAQ and Real-time PCR techniques from the perspectives of serology, histopathology, cell biology and proteomics. The results are as follows: 1. Effects of fluorine on blood routine and ultrastructure of thymus in mice hyperfluorine could inhibit body weight gain and cause damage to thymus ultrastructure in mice. The results showed that nuclear chromatin decreased pyknosis and the membrane structure of double layer membrane was not obvious. Vacuolation of cytoplasm, breakdown of mitochondria ridges and destruction of structure, etc. The results of blood routine examination showed that high fluoride could significantly decrease the number of WBC, lymphocytes, intermediate cells and granulocytes in peripheral blood of mice, and change the proportion of lymphocytes and granulocytes, which indicated that fluoride could affect the immune state of the body. This is consistent with the result of thymus injury. Effects of fluoride on T lymphocyte subsets in mice the changes of T cell subsets in mouse thymus were detected by flow cytometry. The results showed that high dose fluoride could decrease the proportion of CD4 T cells and CD8 T cells in thymus, and the difference was significant. However, the proportion of DP cells in the two fluoride treatment groups increased, while the DN cells decreased in fluoride treatment group, which confirmed that high fluoride could affect the differentiation of thymus T cells and inhibit the production of CD4 or CD8 single positive cells. Effect of fluoride on the interaction between protein and protein in thymus of mice I TRAQ experiment showed that high fluoride induced differential expression of more than 350 proteins. Further bioinformatics analysis showed that these proteins were mainly distributed in more than 110 signal pathways, among which more than 30 involved in cell metabolism, involving more than 70 differential proteins; More than 50 signaling pathways involved more than 160 differential proteins, and 19 pathways were involved in disease development, involving more than 60 proteins. These protein expression disorders can affect the biological process of thymocytes and thus interfere with T cell differentiation. 4. 4. Effect of fluorine on the expression of m RNA, a key gene in T cell differentiation in mice. The results of real-time fluorescence quantitative PCR showed that high fluoride could inhibit the expression of Rps3,Rps3a and Pfn1m RNA, and promote the expression of m RNA of Nefh and Nefm. The results were consistent with the results of protein expression detection. In conclusion, high fluoride can damage the ultrastructure of thymus, affect the differentiation and development of thymus T cells, and induce a large number of differential expression of thymus proteins. From the perspective of proteomics, the effect of high fluoride on the differentiation of T lymphocytes was studied, which laid a foundation for further elucidating the mechanism of fluoride interfering with the differentiation of T cells.
【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R599.1

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