【作者】 周德荣；

【导师】 蔡志明； 桂耀庭；

【作者基本信息】 汕头大学 ， 病理与病理生理学， 2009， 博士

【摘要】 棉酚具有显著的抗男性生育效果,其作为男性避孕药的研究已经进行了将近三十年。国内外对棉酚和有关化合物作为避孕药的作用机理、体内代谢、毒性和临床效果等方面进行了广泛系统的研究,但至今其具体作用机制目前仍不清楚。鉴于以上原因,研究棉酚的男性抗生育机制是男科学一项十分有意义的工作。支持细胞（Sertoli cell）是雄性哺乳动物睾丸曲细精管内唯一的体细胞,可以维持和调节正常的生精过程,在精子发育成熟的过程中具有极为重要的作用。Sertoli细胞的结构和功能的完整性对生精细胞的增殖和成熟十分关键,其改变可导致生精细胞的脱落和死亡。间隙连接（gap junction,GJ）是位于细胞胞膜上连接相邻细胞间的通路,允许小分子及离子物质交换,其主要生化成分为连接蛋白（connexin,Cx）。睾丸中最主要的连接蛋白是connexin43（Cx43）,主要位于曲细精管的基底层Sertoli细胞之间的血睾屏障以及Sertoli细胞和生精细胞之间。近期有多个文献报道,林丹、镉等毒素能减少Sertoli细胞的连接蛋白,并使Sertoli细胞上Cx43的数量减少,从而影响间隙连接通讯（GJIC）的功能,进一步影响到睾丸Sertoli细胞在精子发生过程的正常功能,并对睾丸肿瘤的发生可能也有影响。因此,我们推测棉酚对雄性生殖功能的影响可能也首先从Sertoli细胞开始。本实验的目的是研究棉酚对睾丸支持细胞的毒性作用,检测支持细胞的间隙连接功能,进一步探讨棉酚作用下睾丸支持细胞膜上间隙连接蛋白表达的变化,从而明确棉酚对支持细胞间隙连接的影响。对于研究棉酚的抗生育作用具有重要意义,对于我们进一步开发低毒性男性避孕药有指导意义,同时也可为研究其他环境毒性物质机理提供新思路。在课题的第一部分,采用TM4细胞株,通过CCK-8实验确定棉酚染毒剂量,选定不同浓度（1.25,2.5,5,10μmol/L）棉酚分别处理细胞（6,12,24,48h）,应用划痕标记荧光染料示踪技术检测Sertoli细胞GJIC的功能变化,进一步采用RT-PCR、western blot及细胞免疫荧光法分别从RNA水平、蛋白表达水平及细胞分布变化来探讨棉酚对支持细胞间隙连接的影响。课题的第二部分,改进睾丸支持细胞的分离方法,在体外分离培养小鼠睾丸支持细胞。分别用Feulgen染色法及检测Fas-L表达等方法来鉴定支持细胞的纯度,并用RT-PCR检测棉酚对原代培养的支持细胞上间隙连接的影响。课题第三部分,给成年雄性小鼠胃饲15mg/kg/d棉酚后分别观察4周、8周及12周睾丸的曲细精管,免疫组织化学方法检测支持细胞上间隙连接的变化。实验结果表明,棉酚对TM4细胞有抑制作用。当棉酚浓度＜10^-5mol/L时,细胞存活率大于90%,对TM4细胞的存活率无明显影响;浓度＞5×10^-4mol/L时,对Sertoli细胞存活率的影响随着剂量增大而降低,并呈剂量-效应关系,EC50为1.5×10^-5mol/L。当棉酚浓度为5×10^-3 mol/L时,存活率明显降低,细胞存活率＜3%,其差异有统计学意义（P＜0.05）。5μmol/L棉酚作用TM4支持细胞24小时结果与空白对照组比较,2mm长度内划痕两侧绿色荧光负载细胞数目,空白对照组细胞数为1079.3±290.0,棉酚组为305.6±134.2。组间差异有统计学意义（P＜0.05）,提示棉酚用药后TM4细胞间隙连接功能明显下降。5μmol/L棉酚分别作用TM4支持细胞6小时,12小时,24小时及48小时后RT-PCR结果提示Cx43 mRNA在各组细胞中均有表达,但随棉酚处理时间增加而逐渐下降。与空白组相比,24小时组与48小时组Cx43 mRNA的量均显著下降（P＜0.05）。1.25,2.5,5及10μmol/L棉酚分别处理TM4支持细胞24小时后Westernblot结果提示Cx43蛋白在各剂量组均有表达,但表达量随浓度的增加逐步下降,5μmol/L及10μmol/L组中的表达量均明显低于空白组（P＜0.05）。1.25,2.5及10μmol/L棉酚作用TM4支持细胞24小时后行免疫荧光细胞化学结果可见,棉酚作用组与对照组表达相比较,阳性率和表达强度较正常组均降低,且随剂量增加Cx43蛋白荧光表达强度逐渐减弱。1.25μmol/L及2.5μmol/L组Cx43表达阳性率和表达强度均明显降低,未见有强阳性表达,10μmol/L组只有极少部分为弱阳性表达,位于细胞浆,呈淡绿色斑点状,未见位于细胞膜。在实验的第二部分,成功分离了的原代的小鼠睾丸支持细胞,纯度可达85%以上,RT-PCR分析提示棉酚对其也存在抑制间隙连接蛋白的作用。实验的第三部分,对小鼠进行15 mg/kg/d剂量棉酚处理实验表明,胃饲棉酚4周小鼠睾丸曲细精管的形态无明显改变,但从8周开始,支持细胞膜上的间隙连接蛋白的表达减少,12周时间隙连接蛋白明显减少,提示棉酚对睾丸支持细胞连接蛋白的表达有抑制作用。由此我们得出结论,正常Sertoli细胞存在大量间隙连接通讯,主要成分为Cx43。棉酚能抑制Sertoli细胞的间隙连接通讯功能,而细胞上Cx43表达的减少是其功能受损的物质基础。低剂量棉酚处理小鼠,曲细精管上皮周期频率没有明显紊乱,精子发生过程没有被严重破坏,预示了其作用的可逆性。但可损伤睾丸Sertoli细胞上Cx43,进而干扰影响小鼠睾丸Sertoli细胞的功能,为棉酚导致雄性生殖功能障碍的可能机制之一。更多还原

【Abstract】 The contraceptive effect of gossypol in man had been studied for 30 years. The mechanism and toxicology of gossypol and its allied compound had been widely studied. However, the definite mechanism was still unclear. The study of the reproductive toxicology of gossypol was a very important work in andrology.Sertoli cells were the unique somatocytes in seminiferous of testis. It provided mechanical and nutritional support and played a key role in spermatogenesis. Alterations in Sertoli cell function may lead to a germ cell loss and disruption of the seminiferous epithelium and in turn will lead to impaired spermatogenesis.Gap junctions were composed of intercellular pores that allow the passage of small molecules between adjacent cells. The half channel consisted of a hexamer of specific proteins that belong to the gene family of Connexin. Cx43 was the most important Connexin in the testis, which was localized between Sertoli cells, between Leydig cells, and between Sertoli and germ cells.It was reported that some toxicants such as lindane and cadmium could reduce the connexins in Sertoli cell and block the intercellular communication between cultured human and rat cells. Alterations in gap junctions may be involved in the impairment of spermatogenesis and the pathogenesis of neoplastic seminoma proliferation and then may disrupt the control of germ cell proliferation by Sertoli cells. We presume that the mechanism of suppressing spermatogenesis of gossypol is in the same way.The present study was to investigate the effects of gossypol on Sertoli cell, detect the GJIC on Sertoli cell and approach the mechanism of the Cx43 depression. This would be a significant work in the anti-fertility research of gossypol and would offer a new idea for the studies of the other toxicants. In preliminary experiments, a Sertoli cell line, TM4, was treated with different concentrations of gossypol, 1.25, 2.5, 5 and 10μmol/L for 6, 12, 24 and 48 hours. Cell viability was assessed with CCK-8 assay. GJIC in cells was determined using the scrape loading and dye transfer (SLDT) assay; the expression of Cx43 was detected by RT-PCR, immunofluorescence and Western blot analysis. In the second experiment, we improved the method for Sertoli cells separation from mouse testis in order to obtain more pure cells. Cells were identified by Feulgen staining and FasL detection. RT-PCR was performed to detecte the expression of Cx43. In the third experiment, adult male mice were treated with 15 mg/kg/d gossypol for 4, 8 and 12 weeks. The morphology of the seminiferous epithelium was observed and Cx43 was analyzed by immunohistochemistry.The SLDT assay showed that GJIC between adjacent cells was significantly decreased by gossypol. TM4 cells were incubated with DMSO for 48h or 5μmol/L of gossypol for 6h, 12h, 24h and 48h, respectively. The decrease in Cx43 mRNA occurred as early as 6h after the treatment of gossypol and the effect continued to 48h (P<0.05). Western blot analysis showed that the expression of connexin43 protein was gradually decreased with increasing concentrations when cells were incubated with 1.25, 2.5, 5, or 10μmol/L gossypol for 24 h (P<0.05). The expression of Cx43 was gradually decreased with the increasing concentrations of gossypol, and the effect occurred as early as 6h after the treatment and continued until 48h. The cells were treated with 1.25, 2.5 and 10μmol/L of gossypol for 24 h. The expression of Cx43 was detected by immunofluorescent assay. The intensity of Cx43 immunostaining was dramatically decreased at all doses used (P<0.05). In the second experiment using the combination enzyme digestion method, the purity of Sertoli cells reached above 85%. The RT-PCR assay showed gossypol significantly decreased Cx43 mRNA. In the third experiment, mice were treated with 15 mg/kg/d gossypol, the morphology of the seminiferous epithelium was observed and immunohistochemistry showed that the expression of Cx43 was decreased as early as 8weeks after the treatment and continued until 12weeks.In summary, Cx43 composed gap junctions fundamentally, which were obviously presented between adjacent Sertoli cells. Gossypol could repress the GJIC by decreased the expression of Cx43 and impair the function of Sertoli cells. The seminiferous tubules of mice didn’t show any disorder under the treatment of low dose of gossypol. The results suggested the reversibility of the contraceptive effect of gossypol. However, gossypol could repress the expression of Cx43 in Sertoli cells, which is one of the mechanisms of the reproductive toxicology of gossypol.更多还原