布鲁菌LPS对小鼠子宫内膜蜕膜化的影响

发布时间：2018-02-21 23:44

本文关键词： 布鲁菌LPS 妊娠小鼠子宫内膜蜕膜化　出处：《西北农林科技大学》2015年硕士论文　论文类型：学位论文

【摘要】：布鲁菌脂多糖(lipopolysaccharide,LPS)作为布鲁菌外膜的主要成分,是布鲁菌的毒力因子之一,具有潜在的毒性,而小鼠已被广泛用于探讨LPS诱导妊娠丢失机制的模型建立。子宫内膜蜕膜化是子宫内膜一种特殊的分化过程,在胚胎正常着床和妊娠过程中起重要作用。为了研究布鲁菌脂多糖和妊娠小鼠子宫内膜蜕膜化的关系,本实验拟通过对小鼠子宫内膜蜕膜化组织学观察和相关因子变化的检测,初步探索布鲁菌LPS对妊娠小鼠子宫内膜蜕膜化过程的影响,分析布鲁菌LPS在引起小鼠早期流产中的作用及其机制,为探寻布病防控和检测新靶标提供依据。本试验应用HE染色观察组织形态,利用RT-qPCR、Western Blot和体外分离培养基质细胞及人工诱导蜕膜化等方法检测布鲁菌LPS对妊娠小鼠子宫内膜蜕膜化的影响。试验结果如下:1.以布鲁菌脂多糖作为诱导药物以建立阻碍胚胎着床的模型,将怀孕小鼠分为0、2、5、10μg/g不同剂量LPS组,每组6~8只孕鼠,于孕4 d腹腔注射,孕6 d采样观察。并统计胚胎着床结果。结果显示,随着布鲁菌LPS用药剂量的升高,平均着床胚胎数逐渐降低,当用药剂量为10μg/g时,小鼠胚胎的着床率为0%。5μg/g LPS组的着床率降低,与对照组有极显著差异。因此5μg/g LPS可以作为阻碍小鼠胚胎着床的模型,并用作后续试验。2.应用HE染色显示,与对照组相比,在孕6 d,布鲁菌LPS处理组小鼠子宫蜕膜化区域出现纤维蛋白溶解,在孕8 d,靠近胚胎的蜕膜细胞有大颗粒细胞渗透,蜕膜区域有大量间隙,胚胎周围出现细胞坏死。表明布鲁菌LPS使早期妊娠子宫组织形态发生改变。利用实时荧光定量PCR和Western Blot技术检测布鲁菌LPS对妊娠小鼠子宫内膜蜕膜化相关因子IGFBP-1和CyclinD3表达的影响。结果表明,在孕5~8 d即妊娠小鼠子宫内膜蜕膜化进程中,布鲁菌LPS能够不同程度的抑制小鼠子宫内膜蜕膜化标记因子的表达水平,提示布鲁菌LPS调节小鼠子宫内膜蜕膜化过程,是导致小鼠妊娠失败的原因之一。3.通过分离培养子宫内膜基质细胞及进行体外诱导研究布鲁菌LPS对体外蜕膜化进程的影响。利用实时荧光定量PCR检测布鲁菌LPS对蜕膜化相关因子CyclinD3和dPRP表达的影响,结果表明布鲁菌LPS在不同时间点降低了蜕膜化标志分子的表达,进一步证明布鲁菌LPS能够扰乱小鼠子宫内膜蜕膜化过程,影响小鼠的正常妊娠过程。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.61

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