氟对睾丸支持细胞与淋巴细胞共培养中FasL诱导淋巴细胞凋亡的影响

发布时间：2018-03-16 08:33

本文选题：氟　切入点：支持细胞　出处：《山西农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：[目的]通过染氟支持细胞和淋巴细胞体外共培养后Fas/FasL系统相关基因和蛋白表达的检测,来探讨氟对睾丸支持细胞免疫豁免的毒性机理,从而为支持细胞协助器官移植方面提供理论依据。[方法]本实验设置了不同剂量氟影响下的支持细胞和淋巴细胞共培养,模拟体内睾丸曲细精管中二者的相互作用。通过MTT和TUNEL检测了淋巴细胞的相对存活率和凋亡率；应用QRT-PCR和western blot检测了染氟支持细胞内FasLmRNA和FasL蛋白的表达变化；同时也检测了共培养后淋巴细胞内Fas蛋白的表达和Fas、caspase8和caspase3 mRNA的表达变化。[结果](1)支持细胞鉴定结果：荧光显微镜的蓝光激发下,可见支持细胞相互交织,都像膜一样铺成一层,胞体呈现绿色荧光。计数后可以发现支持细胞的纯度在90%左右,活性达95%以上。可以用来建立细胞模型。(2)氟对淋巴细胞相对存活率影响MTT结果显示：随着氟化钠浓度的上升,淋巴细胞的相对存活率呈现下降趋势,10-6、10-5、10-4mol/L组与对照组淋巴细胞相对存活率相比变化不明显,10-3mol/L组的氟浓度对淋巴细胞相对存活率影响极大,与对照组相比差异极显著。(3)共培养后淋巴细胞相对存活率MTT结果显示：淋巴细胞的相对存活率随着支持细胞攻氟浓度的增加也呈现上升趋势,10-6、10-5mol/L的染氟浓度下,淋巴细胞的相对存活率与对照组相比差异不显著；10-4mol/L组与对照组相比差异显著,10-3mol/L组支持细胞对淋巴细胞相对存活率影响最小,差异极显著。(4)共培养后淋巴细胞凋亡率TUNEL结果：与不同染氟浓度的支持细胞共培养后,淋巴细胞的凋亡率都有所变化,趋势是随着氟化钠浓度的升高而降低,与对照组相比,10-6mol/L组的细胞凋亡率差异不显著(P0.05); 10-5mol/L组与对照组相比凋亡显著(p0.01); 10-4mol/L组差异极显著(p0.01); 10-3mol/L组差异极极显著(5) western blot结果显示：与对照组相比支持细胞各实验组FasL蛋白表达水平有所下降,低剂量(10-5)染氟时,FasL达到显著性差异,10-4和10-3 mol/L的染氟剂量,FasL的表达与对照组相比差异极显著；共培养后淋巴细胞中Fas的表达呈下降趋势,低剂量染氟组淋巴细胞中Fas的表达差异不显著,10-4和10-3mol/L的支持细胞染氟剂量,淋巴细胞中Fas的表达差异极显著。(6) QRT-PCR结果显示：与对照组比,支持细胞FasL mRNA的表达量随着染氟浓度的升高呈现下降趋势；其中10-5mol/L的氟浓度,FasL mRNA的表达无显著性变化；染氟后与对照组组比,10-4、10-5mol/L的氟浓度,FasL mRNA的表达显著升高；共培养后各组淋巴细胞中Fas、caspase3-caspase8三个基因mRNA的表达量与对照组相比,有所下降。其中,Fas、caspase3和caspase8的10-3mol/L氟暴露组与对照组相比,mRNA的表达量均达到显著性差异； Fas和caspase3的10-4mol/L氟暴露组与对照组相比,差异显著；Fas的10-5mol/L氟暴露组与对照组相比,差异显著；[结论]氟通过降低支持细胞FasL基因和蛋白的表达,影响了支持细胞杀伤淋巴细胞的功能,降低了支持细胞的免疫豁免功能。
[Abstract]:[Objective] by detecting expression of Fas/FasL related genes and proteins of fluoride in vitro support cells and lymphocytes after co culture, to explore the mechanism of toxicity of fluoride on Sertoli cell immune privilege, so as to provide the theory basis. The experimental setup method] support cells and lymphocytes of different doses of fluoride under the influence of co culture of Sertoli cells assist in organ transplantation, the interaction in vivo in seminiferous tubules. In two by MTT and TUNEL to detect the relative survival rate and apoptosis rate of lymphocytes; application of QRT-PCR and Western blot to detect the expression changes of fluoride in the supporting cells FasLmRNA and FasL protein; also detected the expression of Fas and Fas protein co culture after the lymphocytes within the expression of caspase8 and Caspase3. The results of mRNA (1)] support the identification of cells: fluorescence excited by blue light, visible support The cells are intertwined, like the film as a paved layer, the cell body showed green fluorescence. After counting Sertoli cells can be found in the purity of about 90%, the activity of more than 95%. Can be used to establish the cell model. (2) fluoride on the relative survival rate of lymphocyte MTT results showed that: with the increase of concentration of sodium fluoride, lymphocyte the relative survival rate decreased, the relative survival rate of 10-6,10-5,10-4mol/L group and the control group did not change significantly compared to lymphocytes, fluoride concentration in 10-3mol/L group the relative survival rate of lymphocyte of great impact, compared with the control group the difference was significant. (3) the relative survival rate of MTT lymphocytes after co culture results showed: with the increase of the relative survival rate Sertoli cells attack fluoride concentration increased lymphocytes, fluoride concentration of 10-6,10-5mol/L lymphocytes, the relative survival rate compared with the control group, the difference was not significant 10-4mol/L; compared with the control group, 10-3mol/L group support cell survival rate with minimal impact on the lymphocyte, the difference was very significant. (4) after the lymphocyte apoptosis rate TUNEL co culture results: co cultured with different concentrations of fluoride in Sertoli cells, lymphocyte apoptosis rate change, the trend is decreased with increasing fluoride the concentration of sodium, compared with the control group, the apoptosis rate of 10-6mol/L group had no significant difference (P0.05); 10-5mol/L group compared with the control group (P0.01); 10-4mol/L apoptosis was significant difference (P0.01); 10-3mol/L group was extremely significant (5) Western blot results showed that: compared with the control cells the experimental group, the expression level of FasL protein decreased, low dose of fluoride (10-5), FasL had significant differences, the fluoride dose 10-4 and 10-3 mol/L, the expression of FasL compared with the control group significantly; The expression of Fas in lymphocytes decreased expression of co cultured, low dose fluoride group Fas in lymphocytes was not significant, and the support of the 10-3mol/L 10-4 cell dose of fluoride, differential expression of Fas in lymphocytes significantly. (6) QRT-PCR results showed: compared with control group, the expression of FasL mRNA cells with the increase of fluoride concentration decreased; fluoride concentration of the 10-5mol/L, no significant changes in the expression of FasL mRNA; fluoride group with the control group, the fluoride concentration of 10-4,10-5mol/L, the expression of FasL mRNA increased significantly after co cultured groups; lymphocyte Fas, caspase3-caspase8 three gene expression of mRNA in with the control group decreased. Among them, Fas, 10-3mol/L Caspase3 and caspase8 fluoride exposed group compared with the control group, the expression of mRNA had significant difference; 10-4mol /L and Caspase3 Fas fluoride exposure Compared with the control group were significantly different; 10-5mol/L Fas fluoride exposed group compared with the control group had significant difference; [Conclusion] fluoride support cells by reducing the expression of FasL gene and protein, the effect of Sertoli cell killer lymphocyte function, decreased immune function from Sertoli cells.

【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R114

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