氟对睾丸支持细胞免疫豁免功能的影响及其机制研究

发布时间：2018-03-20 22:29

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]研究不同浓度氟化钠对小鼠睾丸支持细胞间紧密连接的影响,并对小鼠的睾丸支持细胞进行了体外毒性试验,探究氟对睾丸支持细胞免疫豁免功能的影响,为进一步探讨氟对雄性动物生殖功能的损伤机理奠定基础。[方法]将100只8周龄左右的雄性昆明小鼠随机分为4个试验组(对照组饮用去离子水,低氟组饮用25mg/L NaF的去离子水,中氟组饮用50mg/L NaF的去离子水,高氟组饮用100mg/L NaF的去离子水,饲料均为标准颗粒饲料),攻氟时间为8周,整个试验期间定期记录各试验组小鼠的饮水量及体重。攻毒结束后,计算小鼠的日均摄氟量；测定股氟含量；并摘取睾丸,在电子显微镜下观察睾丸支持细胞的紧密连接。选取出生15-20天左右的雄性小鼠,摘取睾丸,采用酶消化法(胶原酶和胰酶)分离培养睾丸支持细胞,并进行NaF毒性试验,根据毒性试验结果确定体外培养支持细胞攻氟浓度分别为对照组Omol/L,低氟组10-5mol/L,中氟组10-4mol/L,高氟组10-3mol/L,每个剂量组设15个平行样,置于5%C02、37℃培养箱中继续培养48h。攻毒结束后弃去培养液,收集细胞并计数,将106个细胞注射到同种异体小鼠的肾包膜下,术后20天处死动物,取出含移植物的肾脏,常规制备病理切片,采用SABC法检测移植物中蛋白的表达,并用TUNEL技术对移植物中淋巴细胞的凋亡状况进行检测。另外,提取攻毒后支持细胞的总nRNA和总蛋白,采用RT-PCR、Western-blot的方法检测与睾丸支持细胞免疫豁免功能相关的基因与蛋白。[结果]1.各试验组小鼠体重均呈上升趋势,但氟处理组小鼠的体重与对照组相比无显著性差异。股氟含量测定结果显示：低氟组、中氟组和高氟组小鼠股氟含量明显上升,与对照组相比差异显著(P0.05)。2.电镜结果表明：对照组的紧密连接形态完整,呈连续的电子密度较深致密线,且在连结的两侧可看到典型的细胞外质特化区(ES)；低剂量组与对照组相比,变化不明显；中剂量组偶尔会看到很长,但是很细的紧密连接；高剂量组基本观察不到支持细胞间的紧密连接。3.同种异体肾包膜下移植20天后可见在肾包膜下有一白丘状突起,常规切片,HE染色显示肾包膜下有大量的异体细胞,同时肾包膜下移植物的GATA4免疫组化染色证明,有大量阳性棕黄色细胞团聚集在肾包膜下,即为支持细胞。TUNEL法做凋亡观察,可见移植物中有散在的淋巴细胞,胞核棕黄色,且高氟组淋巴细胞凋亡率与对照组先比显著降低。4. RT-PCR结果显示,TGF-β1 mRNA的表达量降低,且在100mg/L氟化钠组较对照组相比差异显著,而25、50mg/L氟化钠组较对照组相比无显著性变化；FasL mRNA的表达量也降低,100mg/L的氟化钠组较对照组相比差异显著；其它相关基因的表达量较对照组相比均无显著性差异。5.Western-blot结果显示与对照组相比各实验组FasL蛋白表达水平呈下降趋势,高氟组的蛋白表达量与对照组相比差异显著；随染氟浓度升高,TGF-β1的蛋白表达水平与对照组相比,高氟组显著,低氟组与中氟组不显著。[结论]试验结果表明：一定浓度的氟会影响睾丸支持细胞的间的紧密连接结构,并且降低睾丸支持细胞的免疫豁免功能。其影响机制与支持细胞内TGF-β1及FasL的蛋白与mRNA表达水平的降低有关,为进一步探讨氟对雄性动物生殖功能的损伤机理奠定理论基础。
[Abstract]:[Objective] to study the effects of different concentrations of sodium fluoride on tight junctions between Sertoli cells of mice, and the mouse Sertoli cells by in vitro toxicity test, explore the influence of fluoride on immune function of Sertoli cell immunity, to further explore the damage mechanism of fluoride on reproductive function of male animal foundation. Method: 100 male Kunming mice were randomly divided into about 8 weeks of age into 4 groups (control group, drinking deionized water, drinking low fluorine group 25mg/L NaF deionized water, fluoride in drinking deionized water group 50mg/L NaF, group 100mg/L NaF high fluoride drinking deionized water, feed are standard pellet feed), fluoride attack time for 8 weeks, during the whole experiment to record water quantity and weight of each experimental group of mice. After inoculation, were calculated daily fluoride intake; Determination of fluorine content and the removal of shares; testis, testis were observed under electron microscope Closely connected pill Sertoli cells. Male mice, born 15-20 days around the testis, using the enzyme digestion method (collagenase and trypsin) cultured Sertoli cells, and NaF toxicity test, according to test results to determine the toxicity of Sertoli cells of fluorine concentration were Omol/L in control group were cultured in vitro, low fluoride group 10-5mol/L. Fluorine in high fluorine group group 10-4mol/L, 10-3mol/L, each dose group of 15 parallel samples, a 5%C02,37 C incubator to culture 48h. challenge after the discard the culture medium, cells were collected and counted, the renal capsule of 106 cells were injected into allogeneic mice, animal were sacrificed 20 days after operation, removed with the graft kidney, routine pathological section was prepared to detect the expression of graft protein by SABC method, and detect the apoptosis of lymphocytes in the graft by using TUNEL technology. In addition, the extraction cell support after virus attack The total nRNA and total protein, using RT-PCR method, Western-blot detection and Sertoli cells immunity function related gene and protein. The weight of each test group]1. mice showed an upward trend, but the fluoride treated mice weight compared with the control group, no significant difference were measured. The results showed that the low fluorine fluorine content shares in the group, the fluoride group and high fluoride group shares fluorine content increased significantly, compared with the control group had significant difference (P0.05).2. electron microscope results showed that the control group is closely connected with morphological integrity, is the continuity of electron density is deep tight line, and even in the junction on both sides can see a typical intracellular region specialization (ES); low dose group compared with the control group, no significant changes in the dose group; occasionally see very long, but closely connected with very small; the high dose group do not observe Sertoli cell tight junctions between.3. allograft renal capsule 20 days after the transplant of a white visible buninoid projection, in the renal capsule of conventional slice, HE staining showed a large number of allogeneic cells under the renal capsule, and GATA4 immunohistochemical staining of the plant under renal capsule that has a large positive Brown cell aggregates in the renal capsule, which support cell.TUNEL method to observe the apoptosis, visible shift in scattered lymphocytes in the plant cell nucleus brown, and apoptosis of lymphocytes in high fluoride group and control group was significantly lower than the first.4. RT-PCR showed that the expression of TGF- beta 1 mRNA decreased, and 100mg/L in the sodium fluoride group than in the control group were significantly higher than those of 25,50mg/L group, and sodium fluoride compared with the control group had no significant change compared with expression of FasL; mRNA also reduced the sodium fluoride group 100mg/L was significantly higher than the control group; the expression of other related genes compared to the control group, there were no significant differences between the.5.Western-blot junction The results show that compared with the control group, the experimental group the expression level of FasL protein decreased, the protein expression of the high fluoride group and control group was significant; with the increasing fluoride concentration increased, TGF- beta 1 expression levels compared with the control group, high fluorine group, low fluorine group and the fluoride group was not significant. Conclusion] test results showed that a certain concentration of fluoride can affect Sertoli cell tight junction structure between, and reduce the function of immune privilege of Sertoli cells. The effect of protein and mRNA mechanisms and support the intracellular TGF- beta 1 and FasL lower expression level, and laid a theoretical foundation for further study on damage mechanism of fluoride the male animal reproductive function.

【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】：S858.91;X503.22

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