【作者】 包图雅；

【导师】 曹贵方；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2010， 博士

【摘要】 防御素是一类富含半胱氨酸的阳离子抗菌肽,它是动物体内防御系统中最大的成员,主要存在于哺乳动物中性粒细胞和上皮细胞中。防御素具有广谱的抗菌,抗病毒等活性,同时,动物防御素还可以调节体内粘膜免疫及其他抗微生物免疫活性。防御素广泛表达在动物组织细胞中,在雌性生殖道中也分布着大量防御素。由于防御素参与天然免疫防御机制,提示防御素可能在雌性生殖系统抵抗微生物感染、维持生殖健康方面起至关重要的作用。生殖道上皮细胞中防御素的表达受到一些激素的调节,特别和雌激素、孕酮有密切的关系。提示雌激素可能参与了对生殖道上皮细胞的防御素表达调节过程,但雌激素是如何实现调控仍不清楚。目前,有关绵羊雌性生殖道防御素和生殖激素的关系的研究极少,也未见生殖激素对绵羊生殖道防御素调控机制的研究。为此,我们初步探讨了雌激素对绵羊输卵管上皮细胞防御素(SBD-1,SBD-2)基因表达的调节作用及其信号通路。结果如下:1、首先建立稳定的绵羊输卵管上皮细胞培养体系。在无菌条件下获取绵羊输卵管上皮细胞,并用0.05%胰酶-0.02%EDTA消化方法,以及差速贴壁法以获取较纯的绵羊输卵管上皮细胞,对绵羊输卵管上皮细胞进行原代培养,以及至少传2代的传代培养。结果显示:经免疫组化方法和形态学方法鉴定,证实所培养细胞具有典型的上皮细胞特征。并且对传2代细胞绘制细胞生长曲线和进行染色体核型分析,证实细胞生长状态良好以及没有发生变异。可以作为下一步研究的实验模型。2、我们首先确定在绵羊输卵管上皮细胞中是否有SBD-1和SBD-2表达,根据已发表的SBD-1和SBD-2序列设计特异性引物,经RT-PCR鉴定及测序证明这两种基因在绵羊输卵管上皮细胞有表达。然后探讨雌激素对绵羊输卵管上皮细胞防御素的表达影响以及是否存在着时间上与剂量上的依赖关系。我们在绵羊输卵管上皮细胞中添加不同剂量(10-6M、10-7M、10-8M、10-9M、10-10 M)17β-雌二醇处理不同时间(2h、6h、12h、24h、48h),然后采用实时荧光定量RT-PCR技术检测雌激素对绵羊输卵管上皮细胞β-防御素(SBD-1,SBD-2)的表达水平。结果表明:17β-雌二醇对绵羊输卵管上皮细胞中SBD-1和SBD-2均存在着明显时间效应和剂量依赖关系,17β-雌二醇在其剂量为10-8M、处理细胞6小时后对SBD-1和SBD-2的上调幅度最大。3、为了进一步明确是何种信号通路介导雌激素对绵羊输卵管上皮细胞防御素(SBD-1,SBD-2)表达的影响,我们采用雌激素核受体阻断剂、膜受体途径可能信号通路阻断剂和GPR30的激动剂处理细胞,然后采用实时荧光定量RT-PCR技术检测SBD-1 mRNA和SBD-2 mRNA的表达变化水平。结果表明:①17β-雌二醇诱导绵羊输卵管上皮细胞中SBD-1和SBD-2基因表达均是由雌激素膜受体途径和雌激素核受体途径共同介导的,并且雌激素核受体途径是主效通路。②17β-雌二醇诱导绵羊输卵管上皮细胞中SBD-1基因表达是由雌激素核受体、PKA、NF-kB和PKC途径所介导的。③17β-雌二醇诱导绵羊输卵管上皮细胞中SBD-2基因表达是由雌激素核受体、NF-kB和PKC途径所介导的。更多还原

【Abstract】 Defensins,a group of cationic antimicrobial peptides contained cysteine-rich,are the biggest component of defense system in creature and mainly exist in heterophil granlocyte and epithelial cell of mammalian.Defensins have broad-spectrum antimicrobial activity,antibacterial activity and so on,at the same time,defensin of animal also regulate the immun of mucous membrane in vivo and other antimicrobial immune activities. In addition,defensins extensively present in animal tissues and cells,and massive defensins are also distribute in female gential tract.Due to defensins participating in innate immune defense mechanism,which indicates that defensins may play essential roles in resisting microbe infection and maintaining reproductive health.The expression of defensin in epithelial cell of gential tract is regulated by certain hormone and relate closely to estrogenic hormone and progestone,which suggests that it is possible for estrogenic hormone to take part in the adjusting progress of defensin expression in epithelial cell of gential tract, but it is unknown to female hormone how to carry out adjusting.Nowadays there is seldom research on the relation between defensin and reproductive hormone in female gential tract of Ovis aries.Thus,the regulation effect of female hormone to the expression of defensin genes (SBD-1,SBD-2) in Uterine tubal epithelium cell and its signal path ways are studied initially in this paper.The results are listed as follows:1. At first,to establish stable culture system of Uterine tubal epithelium cell in Ovis aries.Obtain uterine tubal epithelium cells under germfree condition and then digest cells with 0.05% pancreatic enzyme -0.02% EDTA and centrifuge and adhere cells with different speed,which can lead to gain fair pure uterine tubal epithelium cells,and culture the original generation of uterine tubal epithelium cells,at least the cells culture processing to the second filial generation.The consequence shows that it is confirmed that the cultured cells are epithelial cell through the verification from morphological and immunohistochemical methods.Analyzing chromosomal karyotype and drawing auxodrome curve to the second filial generation cells are confirm cell growth in good condition as well as nonmutation.These cells can be taken as experimental model in future research.2. To determine whether SBD-1 mRNA and SBD-2 mRNA are expressed in sheep oviduct epithelial cells.We designed specific primers based on the published SBD-1 and SBD-2 sequences and then RT-PCR analysis and sequencing revealed that these two genes are expressed in sheep oviduct epithelial cells.Furthermore, to approach the effect of 17β-estradiol onβ-defensin gene expression in the ovine oviduct epithelial cells and whether there to be existed dependent relationship between time and dosage. Sheep oviduct epithelial cells were treated for increasing amounts of time(2h,6h,12h, 24h,48h)with various dose of 17β-estradiol(10-6M、10-7M、10-8M、10-9M、10-10M)and the level ofβ-defensin (SBD-1,SBD-2)gene expression in the ovine oviduct epithelial cells was detected by real-time PCR.The results showed that 17β-estradiol significantly upregulated SBD-1 mRNA and SBD-2 mRNA expression in a time and dose-dependent manner,the maximal stimulatory effect of 17β-estradiol on the expression of SBD-1 mRNA and SBD-2 mRNA were observed at the concentration of 10-8M after treatment for 6h.3. In order to further investigate what kinds of signaling pathway involved mediating the effect of estrogen on the expression of defensins(SBD-1,SBD-2) in sheep oviduct epithelial cells.The changes in the mRNA expression of SBD-1 and SBD-2 in sheep oviduct epithelial cells treated with estrogen nuclear receptor inhibitor and some signaling pathways inhibitors of estrogen membrane receptor and GPR30 agonist were studied by real-time PCR.The results show:①Estrogen nuclear receptor pathway and estrogen membrane receptor pathway may mediate 17β-estradiol induced SBD-1 and SBD-2 genes expression in sheep oviduct epithelial cells,and estrogen nuclear receptor pathway is the main effect.②Multiple pathways such as estrogen nuclear receptor,PKA,NF-κB and PKC may mediate 17β-estradiol induced SBD-1 gene expression in sheep oviduct epithelial cells.③Multiple pathways such as estrogen nuclear receptor, NF-κB and PKC may mediate 17β-estradiol induced SBD-2 gene expression in sheep oviduct epithelial cells.更多还原