高氟干擾T淋巴細胞分化定向蛋白質(zhì)相互作用研究
發(fā)布時間：2019-01-15 21:22
【摘要】：地氟病作為目前我國流行最廣泛,并且嚴重危害到人類健康與動物生命的一種的地方病和多發(fā)病,是由長時間攝入高濃度的氟引起的。大量研究表明,高氟可造成機體的廣泛性損傷,包括免疫系統(tǒng)。本研究通過向小鼠飲水中添加不同劑量的氟(0mg/L,50mg/L,100mg/L)建立動物模型。采用透射電鏡、流式細胞術、i TRAQ和Real-time PCR等技術,從血清學,組織病理形態(tài)學,細胞生物學和蛋白組學等角度研究了高氟的免疫毒性作用。具體結(jié)果如下:1.氟對小鼠血常規(guī)和胸腺超微結(jié)構(gòu)的影響高氟可抑制小鼠的體增重,并造成胸腺超微結(jié)構(gòu)的損傷,表現(xiàn)為核染色質(zhì)減少、固縮,細胞膜核膜的雙層膜結(jié)構(gòu)不明顯,細胞質(zhì)濃縮出現(xiàn)空泡化,線粒體脊斷裂、結(jié)構(gòu)破壞等。血常規(guī)檢查結(jié)果顯示高氟可導致小鼠的外周血WBC數(shù)、淋巴細胞數(shù)、中間細胞數(shù)和粒細胞數(shù)目顯著下降,改變淋巴細胞及粒細胞的比例,表明氟可以影響機體的免疫狀態(tài),這與胸腺損傷的結(jié)果相一致。2.氟對小鼠T細胞亞群的的影響采用流式細胞術檢測小鼠胸腺T細胞亞群的變化,結(jié)果顯示,高劑量的氟可導致胸腺CD4+T細胞和CD8+T細胞的比例下降,且差異顯著,而DP細胞在兩個氟處理組內(nèi)的比例上升,DN細胞在氟處理組中有下降趨勢,證實了高氟可以影響胸腺T細胞的分化,抑制了CD4+或CD8+單陽性細胞的生成。3.氟對小鼠胸腺蛋白質(zhì)與蛋白質(zhì)相互作用的影響i TRAQ實驗結(jié)果顯示,高氟誘導350多個蛋白發(fā)生差異表達。進一步的生物信息學分析表明,這些蛋白主要分布于110多個信號通路,其中參與細胞代謝的有30多條,涉及70多個差異蛋白;參與信號調(diào)控通路的有50多,涉及160多個差異蛋白,參與疾病發(fā)生的有19條通路,涉及60多個蛋白。這些蛋白表達的紊亂可以影響胸腺細胞的生物過程,進而干擾T細胞的分化。4.氟對小鼠T細胞分化關鍵基因m RNA表達的影響實時熒光定量PCR技術驗證差異蛋白m RNA表達影響的結(jié)果顯示,高氟可抑制Rps3、Rps3a及Pfn1m RNA的表達,并促進Nefh和Nefm的m RNA表達,與蛋白表達檢測結(jié)果相一致。綜上所述,高氟可以造成胸腺超微結(jié)構(gòu)的損傷,并影響胸腺T細胞分化發(fā)育,引起大量胸腺內(nèi)蛋白的差異表達。本試驗從蛋白組學的角度出發(fā),研究了高氟對T淋巴細胞分化的影響,為進一步闡明氟干擾T細胞分化定向的機制奠定了基礎。
[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.
【學位授予單位】：河南科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R599.1
[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.
【學位授予單位】：河南科技大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R599.1
【共引文獻】
相關期刊論文 前7條
1 柴麗紅;王宏元;吳民耀;王文科;;氟對不同發(fā)育時期中華大蟾蜍蝌蚪的急性毒性研究[J];安全與環(huán)境學報;2013年04期
2 何綱;丁佩佩;甄沛林;李秀娟;伍金華;湯志強;胡長征;吳興柳;陳曉華;;非耐藥與耐多藥肺結(jié)核患者外周血T淋巴細胞亞群及細胞因子的變化[J];中國感染控制雜志;2013年05期
3 王燕飛;陽益萍;覃麗琳;溫平鏡;韋小敏;;氟對接觸工人外周血淋巴細胞的遺傳毒性[J];工業(yè)衛(wèi)生與職業(yè)病;2014年03期
4 Tingting Yan;Lili Tan;Bingchun Zhang;Ke Yang;;Fluoride Conversion Coating on Biodegradable AZ31B Magnesium Alloy[J];Journal of Materials Science & Technology;2014年07期
5 趙靜;王宏偉;田二杰;周變?nèi)A;;蛋白質(zhì)組學實驗技術及其應用[J];動物醫(yī)學進展;2015年01期
6 呂俊鳥;鄭良s，
本文編號：2409134
本文鏈接：http://sikaile.net/yixuelunwen/nfm/2409134.html
最近更新
教材專著
