【作者】 张晓燕；

【导师】 王俊东；

【作者基本信息】 山西农业大学 ， 临床兽医学， 2017， 硕士

【摘要】 [目的]睾氟升高作为一个氟对生殖系统影响的指标已经被广泛接受,我们推测氟可以通过血睾屏障(Blood testis barrier,BTB),然而,对于氟如何突破BTB导致睾氟含量升高,未进行深入研究。因此,本试验在前期氟致雄性动物生殖功能损伤的基础上,选取大鼠睾丸支持细胞为研究对象,通过建立支持细胞BTB体外模型,针对支持细胞BTB的三种细胞连接方式(TJ、GJ、basal ES),检测氟对支持细胞BTB内稳态的影响及相关分子的变化。从而进一步揭示氟的生殖毒性机理,为实施科学诊断和靶标药物设计提供依据。[方法]选取21天左右的SD雄性大鼠,体外建立原代支持细胞BTB模型,模拟BTB体内系统,于不同浓度NaF体外染毒后,采用XTT法检测细胞活性;应用细胞电阻仪测定TER评价紧密连接屏障功能,同时,通过光漂白荧光恢复技术(FRAP)检测缝隙连接通讯功能;采用qRT-PCR、Western Blotting及IF技术,定量分析三种细胞连接相关基因的表达,以及定量定位分析TJ、GJ以及basal ES相关蛋白的表达情况。[结果]1.本实验成功培养了原代支持细胞,利用HE染色,GATA4和WT1免疫荧光技术鉴定所培养的细胞为纯度极高的支持细胞。2.XTT结果显示,与对照组相比,0.01mg/LNaF对支持细胞有极显著的促进作用(P<0.01);20mg/LNaF对支持细胞有非常显著的抑制作用(P<0.001)。说明低浓度的NaF对细胞的活性有促进作用,高浓度的NaF对细胞的活性有抑制作用。3.紧密连接功能和相关基因蛋白的表达情况:TER的测定结果显示,我们成功构建了体外支持细胞血睾屏障模型。支持细胞染氟(0、0.01、1、10mg/L)48后,与对照组相比,0.1mg/L NaF组电阻值显著升高,10mg/L NaF组电阻值极显著降低,表明0.1mg/LNaF使支持细胞紧密连接功能增强,10mg/LNaF使支持细胞紧密连接屏障功能破坏。qRT-PCR结果显示,各处理组CAR的mRNA表达量与对照组相比无统计学意义;与对照组相比,Occludin的mRNA表达量仅在10mg/L处理组出现显著降低(P<0.05);然而1mg/L处理组的ZO-1与对照组相比,mRNA的表达量上升非常显著(P<0.001)。Western-blot结果显示:与对照组相比,0.1mg/L处理组ZO-1蛋白表达量极显著升高(P<0.01);1mg/L处理组Occludin蛋白表达量显著升高(P<0.05),而10mg/L处理组Occludin蛋白表达量呈极显著下降(P<0.01)。IF技术检测Occludin蛋白的定位情况,对照组和0.1 mg/L处理组支持细胞Occludin蛋白主要在细胞膜上表达,而1 mg/L处理组支持细胞Occludin蛋白主要在靠近细胞核的附近表达,且表达量上升,10mg/L处理组支持细胞Occludin蛋白表达量所有降低。4..basalES相关基因和蛋白的表达情况:与对照组相比,N-Cadherin和α-catenin的mRNA表达量仅在10 mg/L处理组出现显著下降(P<0.05),而β-catenin的mRNA表达量在1 mg/L出现显著升高(P<0.05)。Western-blot结果显示,各处理组α-catenin和β-catenin蛋白表达量与对照组相比无统计学意义,1mg/L处理组N-Cadherin蛋白表达量与对照组相比显著上升(P<0.05)。5.缝隙连接功能和相关基因蛋白的表达情况:与对照组相比,各处理组被漂白的支持细胞荧光恢复值均降低,表明缝隙连接功能被破坏。10mg/L处理组的connexin-43与对照组相比,mRNA的表达量显著下降(P<0.05)。Western-blot结果显示,各处理组connexin-43的蛋白表达量与对照组相比显著降低(P<0.01,P<0.001)。[结论]本试验条件下,NaF暴露后基底胞质外特化相关基因和蛋白未改变;1Omg/L NaF暴露后引起紧密连接屏障功能的破坏,同时下调了 Occludin基因和蛋白的表达;各NaF处理组均导致缝隙连接功能障碍,以及主要蛋白connexin-43表达的下降,所以我们推测缝隙连接是氟诱导血睾屏障结构和功能破坏最重要的靶点。更多还原

【Abstract】 [objective]The increase of fluoride in testis as an indicator of the effect of fluoride on the reproductive system has been widely accepted,so we speculate that the fluorine can pass through the blood testis barrier(BTB),however,that how the breakout of the BTB lead to elevated levels of fluoride in testis is unknown.In this study,the rat testicular sertoli cells were selected as object of study,and the three kinds of cell connections(TJ,GJ,Basal ES)was detect to figure out the effect of fluoride on the homeostasis of BTB and the changes of related molecules in sertoli cells.We aim to further reveal the mechanism of reproductive toxicity of fluoride,and provide the basis for the implementation of scientific diagnosis and target drugdesign.[Method]The BTB model of primary sertoli cells was established in vitro,simulating BTB system in vivo.The cell activity was detected by XTT method after exposure to different concentrations of NaF.The TER was used to evaluate the tight junction barrier,the gap junction communication function detected by light bleach fluorescence recovery technique(FRAP).The expression of three cell connection-related genes was quantitatively analyzed by qRT-PCR,Western Blotting and IF techniques,and the quantitative analysis and positioning analysis of TJ,GJ and basal ES-related proteins were also performed,then to evaluate the effect of fluoride on the structure and function of BTB cells.[Results]1.The sertoli cells were successfully cultured in this experiment.The cultured cells were identified by HE staining,GATA4 and WT1 immunofluorescence,and the purity was very high..2.The results of XTT showed that NaF had a significant effect on the sertoli cells in the 0.00 1mg/LNaF group compared with the control group(P<0.01).Compared with the control group,the 20mg/LNaF treatment group had significant effect on the sertoli cells significant inhibitory effect(P<0.001).Indicating that low concentration of NaF on cell activity can promote the role of high concentrations of NaF on cell activity3.Expression of tight junctions and related gene proteins:The results of TER showed that we successfully constructed a blood testosterone model in vitro.After 48 hour of NaF,the resistance value of 10 mg/L fluoride group significantly decrease compared with the control group,indicating that the sertoli cell tight junction barrier function was destroyed.The expression of CAR mRNA in each treatment group was not statistically significant compared with the control group.Compared with the control group,the expression of occludin and F11 mRNA was significantly decreased in the treatment group(P<0.05).However,the expression of mRNA in the lmg/L treatment group was significantly higher than that in the control group(P<0.001).Western blot results showed that the expression of ZO-1 protein was significantly increased in 0.1 mg/L treatment group compared with the control group(P<0.01).Compared with the control group,the expression of Occludin protein was significantly higher in the 1 mg/L treatment group(P<0.05),while the expression of Occludin protein was significantly decreased in 10mg/LNaF treatment group(P<0.01).The expression of occludin protein in the control group and 0.1 mg/L treatment group was mainly expressed on the cell membrane,while the 1 mg/L treatment group occludin protein was expressed in the vicinity of the nucleus,and the expression level The expression of Occludin was significantly decreased in 10 mg/L treatment group.4.The expression of basal ES related genes and proteins:Compared with the control group,the mRNA expression of N-cadherin and a-catenin was significantly decreased only in the 10mg/L treatment group(P<0.05),but compared with the control group(P<0.05).The expression of β-catenin mRNA was significantly increased at 1 mg/L(P<0.05).Western-blot showed that the expression of a-catenin andβ-catenin protein in each treatment group was not statistically significant compared with the control group.The expression of N-cadherin protein in lmg/L treatment group was significantly higher than that in the control group(P<0.05).5.The expression of gap junctions and related gene proteins were significantly lower in the 1 mg/L and 10 mg/L treated groups than those in the control group,indicating that the gap junction function was destroyed.The expression of connexin-43 in the 10 mg/L treatment group was significantly lower than that in the control group(P<0.05).Western-blot results showed that the protein expression of connexin-43 in each treatment group was significantly lower than that in the control group(P<0.01,P<0.001).IF results showed that the expression of connexin-43 protein in each treatment group disappeared from the cell membrane.[Conclusion]Under the experimental conditions,the effect of fluoride on the basal ES was almost no obvious effect;10mg/L NaF lead to break of tight junction barrier function,and the expression of occludin gene and protein was also decreased under the action of fluoride;the expression of connexin-43 gene and protein in the sertoli cells was decreased,and the communication function of gap junction was also destroyed.it was shown that the most important target for the destruction of BTB in the sertoli cells was the gap junction.更多还原