| 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.
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