本文選題：氟　切入點(diǎn)：支持細(xì)胞　出處：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]研究不同濃度氟化鈉對(duì)小鼠睪丸支持細(xì)胞間緊密連接的影響,并對(duì)小鼠的睪丸支持細(xì)胞進(jìn)行了體外毒性試驗(yàn),探究氟對(duì)睪丸支持細(xì)胞免疫豁免功能的影響,為進(jìn)一步探討氟對(duì)雄性動(dòng)物生殖功能的損傷機(jī)理奠定基礎(chǔ)。[方法]將100只8周齡左右的雄性昆明小鼠隨機(jī)分為4個(gè)試驗(yàn)組(對(duì)照組飲用去離子水,低氟組飲用25mg/L NaF的去離子水,中氟組飲用50mg/L NaF的去離子水,高氟組飲用100mg/L NaF的去離子水,飼料均為標(biāo)準(zhǔn)顆粒飼料),攻氟時(shí)間為8周,整個(gè)試驗(yàn)期間定期記錄各試驗(yàn)組小鼠的飲水量及體重。攻毒結(jié)束后,計(jì)算小鼠的日均攝氟量；測定股氟含量；并摘取睪丸,在電子顯微鏡下觀察睪丸支持細(xì)胞的緊密連接。選取出生15-20天左右的雄性小鼠,摘取睪丸,采用酶消化法(膠原酶和胰酶)分離培養(yǎng)睪丸支持細(xì)胞,并進(jìn)行NaF毒性試驗(yàn),根據(jù)毒性試驗(yàn)結(jié)果確定體外培養(yǎng)支持細(xì)胞攻氟濃度分別為對(duì)照組Omol/L,低氟組10-5mol/L,中氟組10-4mol/L,高氟組10-3mol/L,每個(gè)劑量組設(shè)15個(gè)平行樣,置于5%C02、37℃培養(yǎng)箱中繼續(xù)培養(yǎng)48h。攻毒結(jié)束后棄去培養(yǎng)液,收集細(xì)胞并計(jì)數(shù),將106個(gè)細(xì)胞注射到同種異體小鼠的腎包膜下,術(shù)后20天處死動(dòng)物,取出含移植物的腎臟,常規(guī)制備病理切片,采用SABC法檢測移植物中蛋白的表達(dá),并用TUNEL技術(shù)對(duì)移植物中淋巴細(xì)胞的凋亡狀況進(jìn)行檢測。另外,提取攻毒后支持細(xì)胞的總nRNA和總蛋白,采用RT-PCR、Western-blot的方法檢測與睪丸支持細(xì)胞免疫豁免功能相關(guān)的基因與蛋白。[結(jié)果]1.各試驗(yàn)組小鼠體重均呈上升趨勢,但氟處理組小鼠的體重與對(duì)照組相比無顯著性差異。股氟含量測定結(jié)果顯示：低氟組、中氟組和高氟組小鼠股氟含量明顯上升,與對(duì)照組相比差異顯著(P0.05)。2.電鏡結(jié)果表明：對(duì)照組的緊密連接形態(tài)完整,呈連續(xù)的電子密度較深致密線,且在連結(jié)的兩側(cè)可看到典型的細(xì)胞外質(zhì)特化區(qū)(ES)；低劑量組與對(duì)照組相比,變化不明顯；中劑量組偶爾會(huì)看到很長,但是很細(xì)的緊密連接；高劑量組基本觀察不到支持細(xì)胞間的緊密連接。3.同種異體腎包膜下移植20天后可見在腎包膜下有一白丘狀突起,常規(guī)切片,HE染色顯示腎包膜下有大量的異體細(xì)胞,同時(shí)腎包膜下移植物的GATA4免疫組化染色證明,有大量陽性棕黃色細(xì)胞團(tuán)聚集在腎包膜下,即為支持細(xì)胞。TUNEL法做凋亡觀察,可見移植物中有散在的淋巴細(xì)胞,胞核棕黃色,且高氟組淋巴細(xì)胞凋亡率與對(duì)照組先比顯著降低。4. RT-PCR結(jié)果顯示,TGF-β1 mRNA的表達(dá)量降低,且在100mg/L氟化鈉組較對(duì)照組相比差異顯著,而25、50mg/L氟化鈉組較對(duì)照組相比無顯著性變化；FasL mRNA的表達(dá)量也降低,100mg/L的氟化鈉組較對(duì)照組相比差異顯著；其它相關(guān)基因的表達(dá)量較對(duì)照組相比均無顯著性差異。5.Western-blot結(jié)果顯示與對(duì)照組相比各實(shí)驗(yàn)組FasL蛋白表達(dá)水平呈下降趨勢,高氟組的蛋白表達(dá)量與對(duì)照組相比差異顯著；隨染氟濃度升高,TGF-β1的蛋白表達(dá)水平與對(duì)照組相比,高氟組顯著,低氟組與中氟組不顯著。[結(jié)論]試驗(yàn)結(jié)果表明：一定濃度的氟會(huì)影響睪丸支持細(xì)胞的間的緊密連接結(jié)構(gòu),并且降低睪丸支持細(xì)胞的免疫豁免功能。其影響機(jī)制與支持細(xì)胞內(nèi)TGF-β1及FasL的蛋白與mRNA表達(dá)水平的降低有關(guān),為進(jìn)一步探討氟對(duì)雄性動(dòng)物生殖功能的損傷機(jī)理奠定理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.91;X503.22

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