本文選題：奶牛 + 子宮內(nèi)膜上皮細胞�。� 參考：《華中農(nóng)業(yè)大學》2015年碩士論文

【摘要】：研究表明,在胚胎附植過程中,胎盤完全建立之前,子宮局部呈現(xiàn)缺氧狀態(tài),缺氧造成的子宮內(nèi)環(huán)境的變化可影響多種基因的表達,從而影響著床。本實驗室前期研究已證明缺氧可影響奶牛胚胎關(guān)鍵識別基因BOLA-A的表達。粘蛋白1(MUC1)存在于卵巢、乳腺、胰腺、子宮等多種上皮組織及器官中,是一種跨膜蛋白,分子量高,糖基化水平高。在細胞信號轉(zhuǎn)導中起重要作用,并且能夠調(diào)節(jié)細胞黏附,潤滑上皮組織,在上皮組織的保護中起到一定作用。研究顯示,在人的生殖周期中,子宮內(nèi)膜上皮中的MUC1表達呈一定規(guī)律性,并在分泌中期(著床窗口期)表達量最高。著床窗口期的MUC1表達量與著床率存在一定關(guān)系,可作為子宮容受性標志之一。S100A11是鈣結(jié)合蛋白S100家族中的成員,在腎、肺、皮膚和胎盤等多種組織中均有表達,調(diào)節(jié)以鈣離子為信使的細胞信號轉(zhuǎn)導,與細胞周期及凋亡、炎癥疾病、癌癥等均有相關(guān)。在人類的研究顯示,S100A11存在于子宮的腺上皮和腔上皮,分泌中期表達量高,并且這一時期的低表達與低著床率及不明原因流產(chǎn)存在一定的聯(lián)系。在人類研究中,以上兩個基因?qū)θ焉锲痍P(guān)鍵作用的時期均為著床窗期,而這一時期母胎界面處于相對缺氧狀態(tài)。MUC1與S100A11在奶牛子宮上皮細胞中的表達與否以及缺氧是否對奶牛子宮上皮細胞中二者的表達造成影響是本實驗擬解決的科學問題。本實驗以體外培養(yǎng)4代的奶牛子宮內(nèi)膜上皮細胞為實驗材料,利用本實驗室前期建立起的Co Cl2缺氧模型,通過RT-PCR與Western Blot技術(shù),在m RNA和蛋白水平檢測MUC1與S100A11的表達情況,從而進一步了解缺氧對妊娠調(diào)控起到的作用。研究結(jié)果顯示:奶牛子宮內(nèi)膜上皮細胞表達MUC1和S100A11基因,用不同濃度(低劑量組:100 umol/L;中劑量組:200 umol/L;高劑量組:400 umol/L)Co Cl2處理細胞造成缺氧后發(fā)現(xiàn)在3h后中、高劑量組MUC1基因水平明顯上調(diào),6h及12h后低、中、高劑量組m RNA水平均顯著高于對照組,且高劑量組基因表達結(jié)果明顯高于中、低劑量組。S100A11基因在不同濃度的二氯化鈷刺激下,各時間點m RNA表達水平與對照組相比均顯著上調(diào),并且12h后高劑量組結(jié)果顯著高于中、低劑量組。不同濃度的二氯化鈷處理奶牛子宮內(nèi)膜上皮細胞3h和6h后,各組MUC1和S100A11蛋白水平與對照組無顯著差異。12h后,檢測各組蛋白水平的變化,結(jié)果表明高劑量組MUC1表達水平顯著高于其他組,其余各組間無明顯差異。S100A11在12h后各組蛋白表達水平均無統(tǒng)計學差異。結(jié)果表明,缺氧均在m RNA水平顯著提高奶牛子宮內(nèi)膜上皮細胞表達MUC1和S100A11,且呈現(xiàn)顯著的劑效和時效關(guān)系,且MUC1高劑量12h高劑量組蛋白表達顯著上調(diào)。推測MUC1可能參與了奶牛胚胎著床期子宮內(nèi)環(huán)境的構(gòu)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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.3

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