二硫化碳对共培养大鼠睾丸支持—生精细胞凋亡和凋亡相关蛋白表达的影响

发布时间：2017-12-31 18:20

本文关键词：二硫化碳对共培养大鼠睾丸支持—生精细胞凋亡和凋亡相关蛋白表达的影响　出处：《华中科技大学》2008年硕士论文　论文类型：学位论文

【摘要】： 二硫化碳(CS2)是一种应用广泛的有机溶剂。对男(雄)性影响的报道主要表现为性功能由亢进到减退,精子数减少,活动力下降和畸形精子数增多等。但有关CS2对男(雄)性生殖损伤作用机制的研究鲜有报道。本实验以共培养大鼠睾丸支持-生精细胞为研究对象,初步探讨CS2对大鼠睾丸细胞凋亡作用和凋亡相关蛋白表达的影响,为揭示其基因调控机制,阐明CS2生殖毒性的作用机制提供科学依据。本实验通过建立大鼠睾丸支持-生精细胞共培养体系,运用Feulgen氏核染色法对支持细胞和生精细胞进行鉴定,然后以CS2作为受试物,对共培养大鼠支持-生精细胞进行染毒,用MTT比色法检测其对共培养细胞存活率的影响。用TUNEL法检测支持细胞和生精细胞的凋亡指数,并用Western blot方法对不同浓度CS2处理过的共培养细胞P53和Bcl-2蛋白进行半定量检测,用免疫细胞化学方法对不同浓度CS2处理过的共培养细胞Fas和FasL蛋白进行定位以及粗定量。实验结果表明,本实验方法建立的大鼠睾丸支持-生精细胞共培养体系,在不添加任何细胞因子的条件下,生精细胞能够贴附于支持细胞层上生长,并在体外较长时间共生。在CS2作用下,细胞相对存活率随染毒浓度的增高而降低,当CS2浓度≥100μmol/L时,共培养的支持细胞与生精细胞相对存活率明显降低(P0.05)。凋亡指数亦随染毒浓度的增高而增大(P0.05和P0.01)。P53和Bcl-2蛋白表达水平均随CS2染毒浓度增高而增高,各CS2浓度组P53蛋白表达水平均显著高于对照组(P0.05),100μmol/L和200μmol/L浓度组Bcl-2蛋白表达水平也显著高于对照组(P0.05)。各CS2浓度组Fas和FasL的表达量较对照组明显增高。从以上结果可以看出:在本实验建立的体外共培养体系中,生精细胞可以贴附与支持细胞层上生长,并与之较长时间在体外共生,Feulgen氏核染色法用于鉴定支持细胞和生精细胞简单、易行,我们认为这个培养体系和鉴定方法为深入研究化学物质对支持细胞和生精细胞的生殖毒性及毒作用机制提供了细胞模型;CS2对体外共培养的支持-生精细胞具有细胞毒作用,可使细胞存活率下降,细胞凋亡率增高,最终导致精子数量减少;CS2可诱导共培养大鼠支持-生精细胞P53、Bcl-2、Fas及FasL的表达,Fas和FasL表达上调,启动Fas系统使生精细胞发生凋亡,P53的表达增高进一步促使凋亡的发生,而Bcl-2的高表达可能是为抑制凋亡的发生而产生的保护性反应。因此P53、Bcl-2和Fas系统参与了CS2诱导支持细胞和生精细胞凋亡的基因调控过程,这为进一步探讨其基因调控机制,阐明CS2致男(雄)性生殖损伤的机理,保护职业人群的健康提供了科学依据。
[Abstract]:Carbon disulfide (CS2) is a widely used organic solvent. The effect of CS2 on male (male) sex is mainly characterized by sexual function from hyperactivity to decline, and the number of spermatozoa decreases. However, there are few reports on the mechanism of male (male) reproductive injury caused by CS2. In this study, Sertoli and spermatogenic cells of rat testis were co-cultured. To explore the effect of CS2 on apoptosis and expression of apoptosis-related protein in rat testicular cells, and to provide scientific basis for revealing the mechanism of gene regulation and elucidating the mechanism of reproductive toxicity of CS2. In this experiment, the Sertoli cells and spermatogenic cells of rat testis were identified by Feulgen's nuclear staining method, and then CS2 was used as the test material through the establishment of rat testicular Sertoli and spermatogenic cells co-culture system. The survival rate of co-cultured rat spermatogenic cells was determined by MTT colorimetry, and the apoptosis index of Sertoli cells and spermatogenic cells was detected by TUNEL assay. The p53 and Bcl-2 proteins of co-cultured cells treated with different concentrations of CS2 were detected by Western blot. The Fas and FasL proteins of co-cultured cells treated with different concentrations of CS2 were localized and quantified by immunocytochemistry. The results showed that the spermatogenic cells could grow on the Sertoli cell layer without adding any cytokines in the co-culture system of rat testicular Sertoli cells. Under the action of CS2, the relative survival rate of cells decreased with the increase of the concentration of CS2, when the concentration of CS2 was more than 100 渭 mol/L. The relative survival rate of Sertoli cells and spermatogenic cells in co-cultured Sertoli cells was significantly lower than that in Spermatogenic cells. The apoptosis index also increased with the increase of exposure concentration (P0.05 and P0.01). The expression of p53 and Bcl-2 protein increased with the increase of CS2 concentration. The expression of p53 protein in each CS2 group was significantly higher than that in the control group (P 0.05). The expression of Bcl-2 protein in the 100 渭 mol/L and 200 渭 mol/L groups was also significantly higher than that in the control group (P0.05). The expression of Fas and FasL in each CS2 group was significantly higher than that in the control group. From the above results, we can see that in the co-culture system in vitro, spermatogenic cells can be attached to the Sertoli cell layer and grow with it for a long time in vitro symbiosis. Feulgen's nuclear staining is simple and easy to identify Sertoli cells and spermatogenic cells. We believe that this culture system and identification method provide a cell model for the further study of the reproductive toxicity and toxic mechanism of chemicals to Sertoli cells and spermatogenic cells. CS2 has cytotoxic effect on spermatogenic cells co-cultured in vitro, which can decrease the cell survival rate, increase the apoptosis rate, and eventually lead to a decrease in the number of spermatozoa. CS2 could induce the up-regulation of expression of FAS and FasL in spermatogenic cells of co-cultured rat spermatogenic cells, and induce apoptosis of spermatogenic cells by activating Fas system. The high expression of p53 may promote the occurrence of apoptosis, while the high expression of Bcl-2 may be a protective response to inhibit the occurrence of apoptosis. Bcl-2 and Fas system are involved in the gene regulation of Sertoli cell and spermatogenic cell apoptosis induced by CS2. To elucidate the mechanism of male (male) reproductive injury induced by CS2 and to provide scientific basis for the protection of occupational health.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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