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学科主题: 内科学
题名:
肝性脑病小鼠模型的建立和肝性脑病发病机制的研究
作者: 逄菲
答辩日期: 2014-05-14
导师: 胡瑾华
专业: 内科学
授予单位: 北京大学
授予地点: 北京大学解放军302医院教学医院
学位: 硕士
关键词: 肝性脑病 ; 硫代乙酰胺 ; 小鼠 ; 动物模型 ; 胶质细胞 ; 水通道蛋白4 ; 海马
其他题名: The establishment of mice model and study of the pathogenesis of hepatic encephalopathy
分类号: R747.9
摘要: 目的 1,探索应用硫代乙酰胺(thioacetamide ,TAA)和C57BL/6小鼠制备肝性脑病模型的方法。2,在建立的肝性脑病模型中分别研究脑星形胶质细胞及水通道蛋白-4(AQP-4)在肝性脑病发病中的病理生理改变和作用。 方法 1,建立动物模型 实验分成四组,分别予生理盐水和100、200、300mg/ (kg.d) 剂量的TAA进行腹腔内注射,建立TAA致C57BL/6小鼠肝性脑病的动物模型。注射药物后每24小时,采用旷场实验、ROTA ROD实验及神经功能学评分对小鼠进行神经运动功能的综合评价,观察小鼠自然生存及肝组织病理变化。2,肝性脑病发病机制 实验分为实验组和对照组,对照组给予生理盐水,实验组按照上述实验结果给出的最佳造模方法建立动物模型(200mg/(kg.d ),腹腔注射,连续4 次)。留取实验小鼠脑、肝组织和血清,分别采用免疫荧光染色,western blot,ELISA等方法对小鼠脑海马回内GFAP(胶质纤维酸性蛋白,星形胶质细胞标记蛋白)阳性星形胶质细胞形态变化、水通道蛋白4的表达情况及血清相关炎症因子的变化等进行检测分析,探讨神经胶质细胞水肿、AQP-4,周围血炎症因子在肝性脑病发病中的作用。结果 1,旷场试验,与对照组相比,从造模第1天开始,各实验小组小鼠均出现运动总路程和总时间的明显下降(p <0.05),而各实验组之间无明显差异。 2,ROTA ROD实验,与对照组相比,造模第3天,300mg/ (kg.d)组模型小鼠运动时间明显下降(P =0.036),第4天,各造模小鼠均下降明显(p =0.000),而实验组内无显著性差异。 3,神经功能学评分,造模第4天,200、300mg/ (kg/d)组小鼠的神经功能学评 分平均值分别为6.3和5.1分,与对照组组差异显著(p <0.05)。 4,累积生存率,造模第4天100、200、300mg/ (kg.d)组小鼠的累积生存率分别 为71.43%、71.43%和42.86%。 5,组织学:光学显微镜检查,各实验组小鼠肝组织病理改变符合急性肝衰竭 改变。各实验组小鼠,海马回脑组织结构正常,与对照组比较无差异。 6,GFAP免疫荧光染色,半定量分析结果显示与对照组相比,模型小鼠脑海马CA1区星形胶质细胞胞浆体积明显增加,(t=-2.485,p =0.038),而单位面积内的GFAP阳性细胞计数差异不明显。 7,蛋白质印迹法(western blot) 及其灰度半定量分析结果均显示,与对照组相比,模型组小鼠脑海马区水通道蛋白-4的表达量无明显差异。 结论 1,C57BL/6小鼠和TAA制备的肝性脑病动物模型可用于肝性脑病发病机 制的研究, 200mg/(kg.d) TAA腹腔注射,连续4次,为推荐方法。2,肝性脑病小鼠 模型中,小鼠脑海马CA1区星形胶质细胞水肿,体积增加明显,但水通道蛋白-4 及GFAP的表达量无明显改变。
英文摘要: 【Abstract 】Objective 1,To establish a suitable animal model for the study of hepatic encephalopathy(HE) using thioacetamide(TAA).2,To study the pathogenesis of hepatic encephalopathy involving astrocyte swelling. Methods 1, Establishment of the model: 100 male C57BL/6 mice were devided into four groups. Mice in control group were given normal saline intraperitoneally. Mice in three experiment groups were given an intraperitoneal dose of TAA(100, 200 and 300 mg?kg -1 respectively) . Open field test ,rota rod test, neurological function test and liver pathology were used to evaluate the mice model. 2,The study of pathogenesis:20 male C57BL/6 mice were divided into control group (10) and experimental group (10) which were given a intraperitoneal dose of thioacetamide(TAA( 200mg?kg-1 body weight). Brain, liver and serum sample were collected. Histology changes of brains and livers were detected by H&E staining and protein semi-quantitative calculation.GFAP,an astrocyte marker,was detected by immunofluorescent staining and analyzed with a protein semi-quantitative calculation method.Serum biomarkers of inflammation were tested by ELISA. Results 1. Mice in three experiment groups showed significant shorter total distance and ,no significant difference was observed between themselves. 2. On the third day ,post the initiation of TAA administration,the mean moving time of mice in 300 mg?kg-1group went down significantly in the rota rod test( p =0.036) compared to control group. And all the mice in experiment groups showed significant decline compared with the control group on the fourth day( p =0.000). 3. The average neurological function scores were 9,8.2,6.3 and 5.1 respectively in 100,200,300 mg?kg-1 group on the fourth modeling day. Statistical analysis showed there were significant differences between the control group and both the 200 and 300 mg?kg-1group( p =0.000, p =0.000). 4. The cumulative survival rates of mices in 100,200,300 mg?kg-1 group were 71.43%、 71.43% and 42.86% respectively on the fourth day. 5. Liver pathology: H&E staining of livers exhibited the pathological changes resembling acute liver failure among three experiment groups. 6. The immunofluorescent staining showed significant more GFAP positive cells in modeling group than that of control group( t = -2.485, p =0.038). 7. There was no significant difference in the expressional quantity of aquaporin-4 protein of hippocampus between control group and modeling group ( p =0.855) and AQP-4 expression was assessed by Western blot. 8. Conclusion 1,HE model using C57BL/6 mice induced by TAA appeared to be suitable for the study of the pathogenesis of HE and the dose of TAA were 200 mg?kg-1. 2,The astrocytes located in hippocampus of modeling group were edema. But the difference on the expression content of aquaporin-4,compared with control group,were not observed.
语种: 中文
出处: http://xuewei.bjmu.edu.cn/simpsearch.action?keyword=肝性脑病小鼠模型的建立和肝性脑病发病机制的研究&dbid=72
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内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/105430
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作者单位: 北京大学解放军302医院教学医院

Recommended Citation:
逄菲. 肝性脑病小鼠模型的建立和肝性脑病发病机制的研究[D]. 北京大学解放军302医院教学医院. 北京大学. 2014.
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