缺氧调节奶牛子宫内膜上皮细胞表达MUC1和S100A11的研究

发布时间：2018-06-19 02:35

本文选题：奶牛 + 子宫内膜上皮细胞　；参考：《华中农业大学》2015年硕士论文

【摘要】：研究表明,在胚胎附植过程中,胎盘完全建立之前,子宫局部呈现缺氧状态,缺氧造成的子宫内环境的变化可影响多种基因的表达,从而影响着床。本实验室前期研究已证明缺氧可影响奶牛胚胎关键识别基因BOLA-A的表达。粘蛋白1(MUC1)存在于卵巢、乳腺、胰腺、子宫等多种上皮组织及器官中,是一种跨膜蛋白,分子量高,糖基化水平高。在细胞信号转导中起重要作用,并且能够调节细胞黏附,润滑上皮组织,在上皮组织的保护中起到一定作用。研究显示,在人的生殖周期中,子宫内膜上皮中的MUC1表达呈一定规律性,并在分泌中期(着床窗口期)表达量最高。着床窗口期的MUC1表达量与着床率存在一定关系,可作为子宫容受性标志之一。S100A11是钙结合蛋白S100家族中的成员,在肾、肺、皮肤和胎盘等多种组织中均有表达,调节以钙离子为信使的细胞信号转导,与细胞周期及凋亡、炎症疾病、癌症等均有相关。在人类的研究显示,S100A11存在于子宫的腺上皮和腔上皮,分泌中期表达量高,并且这一时期的低表达与低着床率及不明原因流产存在一定的联系。在人类研究中,以上两个基因对妊娠起关键作用的时期均为着床窗期,而这一时期母胎界面处于相对缺氧状态。MUC1与S100A11在奶牛子宫上皮细胞中的表达与否以及缺氧是否对奶牛子宫上皮细胞中二者的表达造成影响是本实验拟解决的科学问题。本实验以体外培养4代的奶牛子宫内膜上皮细胞为实验材料,利用本实验室前期建立起的Co Cl2缺氧模型,通过RT-PCR与Western Blot技术,在m RNA和蛋白水平检测MUC1与S100A11的表达情况,从而进一步了解缺氧对妊娠调控起到的作用。研究结果显示:奶牛子宫内膜上皮细胞表达MUC1和S100A11基因,用不同浓度(低剂量组:100 umol/L;中剂量组:200 umol/L;高剂量组:400 umol/L)Co Cl2处理细胞造成缺氧后发现在3h后中、高剂量组MUC1基因水平明显上调,6h及12h后低、中、高剂量组m RNA水平均显著高于对照组,且高剂量组基因表达结果明显高于中、低剂量组。S100A11基因在不同浓度的二氯化钴刺激下,各时间点m RNA表达水平与对照组相比均显著上调,并且12h后高剂量组结果显著高于中、低剂量组。不同浓度的二氯化钴处理奶牛子宫内膜上皮细胞3h和6h后,各组MUC1和S100A11蛋白水平与对照组无显著差异。12h后,检测各组蛋白水平的变化,结果表明高剂量组MUC1表达水平显著高于其他组,其余各组间无明显差异。S100A11在12h后各组蛋白表达水平均无统计学差异。结果表明,缺氧均在m RNA水平显著提高奶牛子宫内膜上皮细胞表达MUC1和S100A11,且呈现显著的剂效和时效关系,且MUC1高剂量12h高剂量组蛋白表达显著上调。推测MUC1可能参与了奶牛胚胎着床期子宫内环境的构建。
[Abstract]:The results showed that during embryo implantation, the uterus appeared anoxic state before the placenta was completely established, and the changes of uterine environment caused by hypoxia could affect the expression of many genes and thus the implantation site. Previous studies in our laboratory have shown that hypoxia can affect the expression of the key recognition gene BOLA-A in dairy cattle embryos. Mucin 1 (MUC1) is a transmembrane protein with high molecular weight and glycosylation level, which exists in many kinds of epithelial tissues and organs, such as ovary, mammary gland, pancreas, uterus and so on. It plays an important role in cell signal transduction, and can regulate cell adhesion and lubricate epithelial tissue, and play a certain role in the protection of epithelial tissue. The results showed that the expression of MUC1 in endometrial epithelium was regular in human reproductive cycle and was the highest in the middle of secretory period (implantation window stage). The expression of MUC1 in the window stage is related to the implantation rate, which can be regarded as one of the uterine receptivity markers. S100A11 is a member of the calcium binding protein S100 family. It is expressed in many tissues, such as kidney, lung, skin and placenta. Regulation of cellular signal transduction with calcium ion as messenger is related to cell cycle and apoptosis, inflammatory diseases, cancer and so on. Human studies have shown that S100A11 exists in the glandular and luminal epithelium of the uterus with high expression in the middle secretory phase, and the low expression of S100A11 in this period is related to the low implantation rate and unexplained abortion. In human studies, these two genes play a key role in pregnancy in the period of implantation window, In this period, the expression of MUC1 and S100A11 in the uterine epithelial cells of dairy cattle and the effect of hypoxia on the expression of them in the uterine epithelial cells are the scientific problems to be solved in this experiment. In this experiment, four passages of bovine endometrial epithelial cells were used as experimental materials, and the expression of MUC1 and S100A11 was detected by RT-PCR and Western Blot at the level of mRNA and protein. So as to further understand the role of hypoxia on the regulation of pregnancy. The results showed that MUC1 and S100A11 genes were expressed in the endometrial epithelial cells of dairy cows. The cells were treated with different concentrations of MUC1 and S100A11 (low dose group: 100 umolr / L; middle dose group: 200 umolr / L; high dose group: 400 umol/ L ~ (-1) Co Cl _ 2). After hypoxia, the cells were found in 3 hours after hypoxia. The mRNA level of MUC1 gene in high dose group was significantly higher than that in control group after 6 h and 12 h. The gene expression of high dose group was significantly higher than that of medium dose group, and the gene expression of S100A11 gene in low dose group was stimulated by different concentrations of cobalt dichloride. Compared with the control group, the level of mRNA expression in the high dose group was significantly higher than that in the middle and low dose group at all time points, and the results in the high dose group were significantly higher than those in the middle and low dose group. After treated with cobalt dichloride for 3 h and 6 h, the protein levels of MUC1 and S100A11 in each group were not significantly different from those in the control group. The results showed that the expression level of MUC1 in high dose group was significantly higher than that in other groups, and there was no significant difference in protein expression between other groups after 12 hours. The results showed that hypoxia significantly increased the expression of MUC1 and S100A11 in dairy cow endometrial epithelial cells at the level of mRNA, and showed a significant dose-effect and time-dependent relationship, and the expression of histone in high dose of MUC1 at 12h was significantly up-regulated. It is speculated that MUC1 may be involved in the construction of intrauterine environment during embryo implantation.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.3

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