本文關(guān)鍵詞： 布魯菌LPS 妊娠小鼠 子宮內(nèi)膜 蛻膜化　出處：《西北農(nóng)林科技大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：布魯菌脂多糖(lipopolysaccharide,LPS)作為布魯菌外膜的主要成分,是布魯菌的毒力因子之一,具有潛在的毒性,而小鼠已被廣泛用于探討LPS誘導(dǎo)妊娠丟失機制的模型建立。子宮內(nèi)膜蛻膜化是子宮內(nèi)膜一種特殊的分化過程,在胚胎正常著床和妊娠過程中起重要作用。為了研究布魯菌脂多糖和妊娠小鼠子宮內(nèi)膜蛻膜化的關(guān)系,本實驗擬通過對小鼠子宮內(nèi)膜蛻膜化組織學(xué)觀察和相關(guān)因子變化的檢測,初步探索布魯菌LPS對妊娠小鼠子宮內(nèi)膜蛻膜化過程的影響,分析布魯菌LPS在引起小鼠早期流產(chǎn)中的作用及其機制,為探尋布病防控和檢測新靶標提供依據(jù)。本試驗應(yīng)用HE染色觀察組織形態(tài),利用RT-qPCR、Western Blot和體外分離培養(yǎng)基質(zhì)細胞及人工誘導(dǎo)蛻膜化等方法檢測布魯菌LPS對妊娠小鼠子宮內(nèi)膜蛻膜化的影響。試驗結(jié)果如下:1.以布魯菌脂多糖作為誘導(dǎo)藥物以建立阻礙胚胎著床的模型,將懷孕小鼠分為0、2、5、10μg/g不同劑量LPS組,每組6~8只孕鼠,于孕4 d腹腔注射,孕6 d采樣觀察。并統(tǒng)計胚胎著床結(jié)果。結(jié)果顯示,隨著布魯菌LPS用藥劑量的升高,平均著床胚胎數(shù)逐漸降低,當用藥劑量為10μg/g時,小鼠胚胎的著床率為0%。5μg/g LPS組的著床率降低,與對照組有極顯著差異。因此5μg/g LPS可以作為阻礙小鼠胚胎著床的模型,并用作后續(xù)試驗。2.應(yīng)用HE染色顯示,與對照組相比,在孕6 d,布魯菌LPS處理組小鼠子宮蛻膜化區(qū)域出現(xiàn)纖維蛋白溶解,在孕8 d,靠近胚胎的蛻膜細胞有大顆粒細胞滲透,蛻膜區(qū)域有大量間隙,胚胎周圍出現(xiàn)細胞壞死。表明布魯菌LPS使早期妊娠子宮組織形態(tài)發(fā)生改變。利用實時熒光定量PCR和Western Blot技術(shù)檢測布魯菌LPS對妊娠小鼠子宮內(nèi)膜蛻膜化相關(guān)因子IGFBP-1和CyclinD3表達的影響。結(jié)果表明,在孕5~8 d即妊娠小鼠子宮內(nèi)膜蛻膜化進程中,布魯菌LPS能夠不同程度的抑制小鼠子宮內(nèi)膜蛻膜化標記因子的表達水平,提示布魯菌LPS調(diào)節(jié)小鼠子宮內(nèi)膜蛻膜化過程,是導(dǎo)致小鼠妊娠失敗的原因之一。3.通過分離培養(yǎng)子宮內(nèi)膜基質(zhì)細胞及進行體外誘導(dǎo)研究布魯菌LPS對體外蛻膜化進程的影響。利用實時熒光定量PCR檢測布魯菌LPS對蛻膜化相關(guān)因子CyclinD3和dPRP表達的影響,結(jié)果表明布魯菌LPS在不同時間點降低了蛻膜化標志分子的表達,進一步證明布魯菌LPS能夠擾亂小鼠子宮內(nèi)膜蛻膜化過程,影響小鼠的正常妊娠過程。
[Abstract]:As the main component of brucellosis outer membrane, lipopolysaccharide (LPS) is one of the virulence factors of brucellosis and has potential toxicity. The mouse model has been widely used to study the mechanism of pregnancy loss induced by LPS. Decidualization of endometrium is a special process of endometrial differentiation. In order to study the relationship between brucellosis lipopolysaccharide and decidualization of endometrium of pregnant mice, the purpose of this study was to observe the histological changes of decidualization and the changes of related factors in mouse endometrium. To explore the effect of brucellosis LPS on decidualization of endometrium in pregnant mice, and to analyze the role and mechanism of brucellosis LPS in early abortion of mice. In order to provide the basis for the prevention and control of brucellosis and the detection of new targets, the tissue morphology was observed by HE staining. The effects of Brucella LPS on decidualization of endometrium of pregnant mice were detected by RT-qPCRX Western Blot, isolation of culture medium and artificial induced decidualization in vitro. The results are as follows: 1. Brucella lipopolysaccharide was used as an inducer to induce decidualization of endometrium of pregnant mice. Building models that block embryo implantation, The pregnant mice were divided into 10 渭 g / g LPS group with 10 渭 g / g, 6 pregnant mice in each group were injected intraperitoneally on the 4th day of gestation and observed on the 6th day of gestation. The results of embryo implantation were analyzed. The results showed that the dose of brucellosis LPS increased with the increase of the dose of brucellosis LPS. The average number of implanted embryos decreased gradually, and when the dosage was 10 渭 g / g, the implantation rate of mouse embryos decreased significantly in the 0.5 渭 g / g LPS group, which was significantly different from that in the control group, so 5 渭 g / g LPS could be used as a model to block the implantation of mouse embryos. The results of HE staining showed that fibrinolysis appeared in decidua of mice treated with Brucella LPS at 6 days of gestation, and large granulosa cells infiltrated in decidua cells close to embryo at 8 days of gestation. There are a lot of gaps in the decidua region. It was suggested that brucellosis LPS caused the change of uterine tissue morphology in early pregnancy. Real-time fluorescence quantitative PCR and Western Blot technique were used to detect IGFBP-1 of decidua associated with brucellosis LPS in endometrium of pregnant mice. And the effect of CyclinD3 expression. In the process of decidualization of endometrium in pregnant mice, brucellosis LPS could inhibit the expression of decidualization marker in different degree, suggesting that Brucella LPS regulated the process of decidualization in mouse endometrium. Through isolation and culture of endometrial stromal cells and induction in vitro to study the effect of Brucella LPS on the process of decidualization in vitro. Real time fluorescence quantitative PCR was used to detect the effect of brucella LPS on the process of decidualization in vitro. The expression of decidua-related factors CyclinD3 and dPRP, The results showed that Brucella LPS decreased the expression of decidualization markers at different time points, which further demonstrated that Brucella LPS could disrupt the decidualization process of mouse endometrium and affect the normal pregnancy process of mice.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.61

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