【作者】 赵士光；

【导师】 陈小玉；

【作者基本信息】 郑州大学 ， 卫生毒理学， 2008， 硕士

【摘要】 近年来,环境污染已成为十分突出的问题,重金属是其中一类重要的污染物。铅是人类最早开始使用的重金属元素之一,因其应用广泛,也造成了对环境的污染。铅可对机体多个系统和器官造成损伤,并影响动物和人类的生殖功能。国内外以往关于铅雄性生殖毒性的研究主要集中在睾丸,因为睾丸为雄性最主要的生殖腺,并且是大多数外源化学物雄性生殖毒性的靶器官。支持细胞是雄性动物睾丸曲细精管内唯一的体细胞,在雄性生殖系统中发挥着重要作用。支持细胞离体培养模型由于具有快速、经济、干扰少、实验结果针对性强以及结果的可靠性和重现性好等特点,被广泛应用于外源性毒物的生殖毒理学研究中。支持细胞被称为生精细胞的“保姆细胞”,不仅在结构上为生精细胞的分化成熟提供支架,供给生精过程所需的能量及营养物质:同时可分泌数十种物质参与生精细胞的分化成熟,具有精细的协调作用,保证精子发生的正常有序进行。支持细胞可分泌雄激素结合蛋白（androgen binding protein,ABP）、转铁蛋白（transferrin,Tf）和抑制素（inhibin,INH）等。ABP能够转运和储存雄激素,使曲细精管内维持高浓度的雄激素,生精细胞和精子均有结合和代谢ABP的能力;Tf可以为生精细胞提供生理活动和生化过程所需要的铁,并且对细胞生长有直接的刺激作用;INH能够抑制卵泡刺激素（follicle stimulating hormone,FSH）的分泌,不仅在调节垂体-睾丸轴的平衡中起着必不可少的作用,而且在睾丸精细胞分化、发育过程中起着直接的作用。ABP、Tf和INH均被广泛地用作支持细胞功能的标志。铅能够造成FSH的分泌异常,影响支持细胞内第二信使的浓度,也能够直接作用于支持细胞,影响支持细胞内第二信使的生成。上述变化可能会影响到支持细胞内基因的表达,造成支持细胞分泌功能的异常,产生雄性生殖毒性。本研究通过体外分离培养大鼠睾丸支持细胞,使其暴露于不同浓度的醋酸铅,从mRNA水平上分析受试物对支持细胞表达ABP、INH和Tf的影响。以此探讨支持细胞在醋酸铅造成的雄性生殖功能损伤中的作用,为进一步研究铅的生殖毒性作用提供理论依据。方法:1.建立大鼠睾丸支持细胞体外原代培养模型。18～21日龄SD大鼠睾丸经胰蛋白酶和胶原酶依次消化,分离和纯化支持细胞。在35℃、5%CO₂条件下进行培养,并经Feulgen染色进行支持细胞鉴定。利用MTT法描述支持细胞生长曲线。以DMEM/F-12培养基为溶剂配制醋酸铅工作液,给予不同剂量对支持细胞染毒24h,确定醋酸铅最大无细胞毒性作用剂量。2.醋酸铅对ABP、INH和Tf基因转录的影响。以醋酸铅浓度为0 mol/L、10^-8mol/L、10^-7mol/L、10^-6mol/L和10^-5mol/L对离体原代培养的大鼠睾丸支持细胞染毒24h,提取细胞内总RNA,进行逆转录聚合酶链反应（RT-PCR）,以GAPDH为内参照,检测支持细胞内ABP、INH和TfmRNA的水平。3.统计学分析。运用SPSS 12.0统计软件的单因素方差分析（analysis ofvariance,ANOVA）、Bonfferoni两两比较、Levene方差齐性检验对数据进行统计学分析（检验水准α=0.05）。结果:1.本研究分离培养的支持细胞体外生长良好,且支持细胞纯度比较高,可以用于体外实验研究。不同浓度的醋酸铅染毒体外培养第4d的支持细胞24h后,结果发现醋酸铅浓度最高为10^-5mol/L时无细胞毒性。2.RT-PCR结果:①以10^-8mol/L～10^-5mol/L的醋酸铅染毒支持细胞24h,ABP mRNA相对表达量均高于对照组,但是差异均无统计学意义（P＞0.05）。②以10^-8mol/L～10^-5mol/L的醋酸铅染毒支持细胞24h,TfmRNA在10^-8 mol/L、10^-7mol/L和10^-6 mol/L剂量组的相对表达量均高于对照组,10^-5 mol/L剂量组的相对表达量低于对照组,但是各剂量组的基因相对表达量与对照组比较,差异均无统计学意义（P＞0.05）。③以10^-8mol/L～10^-5mol/L的醋酸铅作用于支持细胞24h后,INH mRNA在10^-8mol/L和10^-7 mol/L剂量组的相对表达量低于对照组,差异具有统计学意义（P＜0.05）。10^-6 mol/L剂量组的相对表达量低于对照组,10^-5mol/L剂量组的相对表达量高于对照组,但是差异均无统计学意义（P＞0.05）。结论:1.醋酸铅对原代培养大鼠睾丸支持细胞在10^-8mol/L～10^-5mol/L剂量范围内未观察到细胞毒性。2.醋酸铅在10^-8mol/L～10^-5mol/L剂量范围内对支持细胞雄激素结合蛋白和转铁蛋白的转录表达无影响,醋酸铅在此剂量下不影响雄激素结合蛋白和转铁蛋白的分泌。3.醋酸铅在10^-8mol/L和10^-7 mol/L剂量下可减少支持细胞抑制素的转录表达。醋酸铅在10^-6 mol/L和10^-5mol/L剂量下对抑制素的转录表达没有影响。醋酸铅可以改变抑制素的分泌,影响抑制素在生殖系统中的作用。更多还原

【Abstract】 In recent years, environmental pollution has become a prominent issue. Heavy metal is one of the most important pollutants. Lead is one kind of the heavy metal that began to use at the early period in the human history. It is applied widely, and it pollutes the environment. Lead has adverse effects on several systems and organs in body. It also can affect the reproductive function of animal and human.The previous studies about male reproductive toxicity of lead mainly focused on testis. Testis is the most important male gonad and the main target organ of most of xenobiotics. The Sertoli cell is the only body cell in the mammalian testicle’s seminiferous tubule. Sertoli cells play a very important role in male reproductive system. Primary culture of Sertoli cells in vitro is characterized by its rapid speed, less cost, less interference, and its results are more target, reliability and reproducibility. It is applied in the study of the reproductive toxicology of xenobiotics.Sertoli cells are called "nursing cell" of spermatogenic cells. Sertoli cells have multiple functions such as providing the cytoarchitecture support and energy and nourishment for developing germ cells. Sertoli cells can also synthesize and secrete many different gene products, which have important biologic functions. Sertoli cells’ gene products include androgen binding protein （ABP）, transferrin （Tf） and inhibin （INH ） and so on. ABP can especially combine with testosterone to maintain a high concentration of androgen in the testicle’s seminiferous tubule, which facilitates the development and maturation of spermatogenic cells. Tf is a major secretory product of Sertoli cells. Tf is postulated to transport Fe³⁺ to the developing germ cells sequestered by the blood-testis barrier and promote spermatogenic cells’s growth and maturation. INH can act on pituitary gonadotropes to suppress FSH secretion. Furthermore, INH appears to have local effects within the testis to suppress spermatogonial numbers. ABP, INH and Tf are so far regarded as the best endocrine marker of Sertoli cells to evaluate spermatogenesis in the testes. Lead can disrupt the secretion of FSH, and this will change the concentration of second messenger in Sertoli cells. Lead can act on Sertoli cells directly and change the concentration of second messenger, too. These changes may disrupt the expression of gene products in Sertoli cells. The abnormity of Sertoli cell’s function may be the mechanism of lead’s toxic effect on male reproductive system.Therefore, the goal of the present study was to determine the effects of different concentration of lead acetate on cell proliferation and expressions of ABP, INH and Tf in primary cultured Sertoli cells isolated from immature Sprague- Dawley rats to explore the molecular mechanisms of reproductive malfunction of male mammal caused by lead acetate.Methods:1. A method was set up for obtaining a large number of viable Sertoli cells from Sprague- Dawley rats of 18～20 days of age. Minced whole testes from SD rats were sequentially treated with 0.25% pancreatin for 15～20 min and 0.1% collagenase for 20～25 min, then washed Sertoli cells sequently, were cultured in DMEM/F-12 media with 10% fetal bovine serum incubated at 35℃in a humidified incubator in an atmosphere of 5% CO₂. On the 4th day of culture, contaminating spermatogenic cells were lysed with a hypotonic solution of 20mM Tris- HCL for 5 min. Feulgen staining had been used to identify the purification and viability of Sertoli cells. Subsequently we established the growth curve of Sertoli cells in vitro and ascertained the maximum dose of non- cytotoxicity of lead acetate on Sertoli cells via MTT method. Sertoli cells were treated with different concentrations of lead acetate for 24h. The concentration of lead acetate in DMEM/F-12 media were 0 mol/L, 10^-9mol/L, 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L.2. The Sertoli cells cultured for 4 days in 100 mL culture bottles were treated with different concentrations of lead acetate and then transferred into the incubator with 5% CO₂ humidified air to continue to incubate for 24h at 35℃. According to the results of Sertoli cells toxicity of lead acetate by MTT method, Sertoli cells exposed to 10^-8 mol/L, 10^-7 mol/L, 10^-6 mol/L and 10^-5 mol/L were used for the total RNA extraction. 0mol/L was taken as control. The total RNA extraction was used in the subsequent reverse transcription. ABP, INH, Tf and GAPDH were amplified by PCR kit. GAPDH was used as control of the quality of the RNAs. The levels of ABP, INH and Tf expressions were measured by densitometric analysis and standardized by comparison to the GAPDH control using a digital imaging and analysis system.3. Statistical analysis results were presented as mean±SD. Statistical comparisons referred to Bonfferoni test for multiple comparisons when significant difference were detected by one- way analysis of variance （ANOVA）. A difference at P<0.05 was considered statistically significant.Results:1. Sertoli cells isolated from rat testis for primary culture grew well in vitro. The viability of Sertoli cells was nonsignificantly reduced at the different concentrations of lead acetate treatment for 24h. The lead acetate didn’t have cytotoxicity at the concentration of 10^-5mol/L.2. The levels of the expression of ABP mRNA at different concentrations were all higher than that of the control group, but the increase were not statiscally significant （P>0.05）. The expression of Tf mRNA increased at the dose of 10^-8 mol/L, 10^-7 mol/L and 10^-6 mol/L while the expression decreased at the dose of 10^-5 mol/L. But these changes compared with control group were not statiscally significant （P>0.05）. The expression of INH mRNA decreased at the dose of 10^-8 mol/L and 10^-7 mol/L, and these changes were statiscally significant （P<0.05）. But the changes at the dose of 10^-6 mol/L and 10^-5 mol/L were not statiscally significant （P>0.05）.Conclusions:1. The lead acetate don’t have cytotoxicity at the concentration from 10^-8 mol/L to 10^-5mol/L.2. The lead acetate has no impact on the expression of ABP mRNA and Tf mRNA at the concentration from 10^-8 mol/L to 10^-5mol/L.3. The lead acetate can decrease the expression of INH mRNA at the concentrations of 10^-8 mol/L and 10^-7 mol/L and has no impact on the expression of INH mRNA at the concentrations of 10^-6 mol/L and 10^-5mol/L.更多还原