Caveolin-1及相关信号通路在甲醛诱导附睾上皮细胞氧化损伤中的作用

Formaldehyde (FA) is a ubiquitous environmental pollutant. Over the last 20 years, China has experienced rapid economic growth and a simultaneous rise in demand for FA. In 2004, China surpassed the United States as the largest FA producer and consumer in the world. China's actual FA output has reached a staggering 12000 kt in 2007, about 4000 times the amount five decades earlier. Coinciding with the growth, FA pollution has also increased considerably in China. .Human reproduction is a delicate process that can be influenced by many factors. Recently, there are growing concerns regarding the toxicity of FA exposure on male reproduction. Epididymis is known to play an important role in the maturation and storage of sperm. Our earlier studies indicated that the epididymis is one of important target organs of FA reproductive toxicity. Histopathological studies showed that atrophy of epididymal tubules; disintegrity, disorganization and even vacuolar-denaturalization of the epididymal epithelial cells in rats of FA exposure. Furthermore, FA exposure alters the epididymal structure and function by inducing oxidative injuries in epididymis of adult rats. However, the underlying molecular mechanism of cellular signal transduction remained to be elucidated. .Caveolin-1 is a major structural component of raft structures within the cell membrane. As the key regulator of cell signal transduction, Caveolin-1 not only participates in many physiological functions of cells, but also extensive involved in pathological processes of celluar oxidative injuries. More recent data addressed that the decreased expression of Caveolin-1 was associated the activation of many signal pathways which involved in pathological processes such as lung, liver, kidney and endothelial oxidative injuries. .Our earlier studies demonstrated that Caveolin-1 is ubiquitously expressed in epididymal epithelial cells. Moreover, our studies first found that the decreased expression of Caveolin-1 was associated with the increasing of FA exposure dose and oxidative stress levels of epididymal tissues. So we speculated that Caveolin-1 plays an important role in epididymal oxidative injuries induced by FA exposure. However, the underlying molecular mechanism of cellular signal transduction is unclear. Therefore, the present study was designed to take Caveolin-1 as the breakthrough point, to investigate the effects of Caveolin-1 and its related signal transduction pathways on epididymal epithelial cells oxidative injuries induced by FA at the molecular, cellular and overall animal levels, by using various methods such as functional proteomics, immunoprecipitation, laser confocal, immunohistochemistry, western blot, real-time RT-PCR, gene transfection and siRNA etc.. This study will not only elucidate the signaling pathways of epididymal epithelial cells, but also lay down a theoretical and experimental basis for prevention and treatment male reproductive toxicity of FA.

在众多的环境污染物中，甲醛以来源广，毒性大，污染时间长等特点已成为全球最主要的环境污染物之一。附睾是精子成熟的重要场所，我们前期的研究显示附睾是甲醛雄性生殖毒性的重要靶器官之一，氧化应激是甲醛引起附睾上皮损伤的重要机制。但此过程中具体的信号转导分子机制仍不清楚。Caveolin-1，是信号整合中心的枢纽，可以和多种信号蛋白相互作用而广泛参与疾病的发生发展过程。本研究将在我们前期广泛进行甲醛雄性生殖毒性和Caveolin-1表达研究的基础上，以Caveolin-1为突破点，运用功能蛋白质组学，免疫共沉淀、激光共聚焦、免疫组化、蛋白印迹、实时动态定量RT-PCR、基因转染和siRNA等多种研究手段，从分子、细胞和动物整体水平综合研究Caveolin-1及相关信号通路在甲醛诱导附睾上皮细胞氧化损伤中的具体作用和机制。本研究将为阐明附睾上皮细胞信号通路奠定基础，为防治甲醛雄性生殖毒性提供理论依据。

在众多的环境污染物中，甲醛以其来源广，毒性大，污染时间长等特点已经成为全球最主要的环境污染物之一。尽管人群研究和动物实验已经显示甲醛具有雄性生殖毒性，但甲醛的致毒分子机制尚不明确。本研究以信号整合中枢蛋白Caveolin-1为切入点，一方面利用RT-RCR, Wetern blot, 免疫组化和免疫荧光等方法观察了Caveolin-1在原代培养大鼠附睾上皮细胞中以及大鼠附睾组织的时空表达，发现Caveolin-1mRNA和蛋白在各年龄段大鼠附睾组织中均有丰富表达。甲醛染毒后大鼠附睾组织Caveolin-1表达下调，且随着甲醛染毒剂量以及附睾组织氧化应激程度的增加其表达进一步下降。这些结果为进一步探索Caveolin-1在生殖系统的功能奠定了实验基础。另一方面，我们利用功能蛋白质组学技术初步筛选鉴定出附睾上皮细胞上与Caveolin-1脚手架区（信号转导区域）相结合的信号蛋白，这些蛋白主要参与（1）细胞的增殖、凋亡与自噬；（2）细胞信号传导；（3）氧化应激反应；（4）膜系统物质转运与细胞骨架的形成；（5）细胞的能量代谢等。其中自噬通路，凋亡通路以及MAPK通路等可能是甲醛雄性生殖毒性的重要通路。进一步，我们利用大鼠甲醛气体吸入染毒实验进一步研究了自噬现象在甲醛雄性生殖毒性中的作用。通过透射电镜技术观察大鼠睾丸组织自噬体的形成，利用Western blot技术观察自噬标志物LC3Ⅰ向LC3Ⅱ的转换，通过RT-PCR技术观察自噬标记物LC3Ⅱ在mRNA水平上的变化，进一步发现一定剂量的甲醛吸入可以促进大鼠睾丸组织自噬现象增加，自噬机制可能是甲醛雄性生殖毒性的重要机制。本课题经过一年的研究，发表相关SCI收录论文2篇，核心期刊论文1篇。培养博士研究生1名，硕士研究生3名。研究结果为甲醛雄性生殖毒性及机制的研究提供了丰富的资料和数据，为预防甲醛生殖毒性提供了实验依据和下一步的研究方向。