Wilson病铜过量负荷动物模型的建立及其肝损伤机制的初步探讨

发布时间：2018-02-26 09:01

本文关键词： WD 肝细胞凋亡 Bax Bcl-2　出处：《华中科技大学》2007年硕士论文　论文类型：学位论文

【摘要】： 【背景及目的】肝豆状核变性(Wilson disease, WD)是常染色体隐性遗传的铜代谢障碍疾病,发病率约为3/10万。WD好发于青少年,其病因是由于ATP7B基因突变导致ATP7B功能出现缺陷,不能清除体内多余的铜,导致铜在肝脏等组织中沉积。临床上以慢性肝脏病变、肾脏功能损害以及神经系统障碍等症状为主。但WD的铜代谢障碍起源于肝脏,主要异常部位也在肝脏,50%以上的病人临床表现为肝功能减退、肝中过多的铜引起的病理改变主要为肝细胞坏死、门静脉及其周围炎症和纤维化。目前对铜沉积在肝脏引起肝脏损伤的具体机制还不明确,国内外的研究表明凋亡在此损伤过程中起着重要的作用,但是其具体的调节机制并不明确。Bcl-2家族调控蛋白比例的失衡在多种原因引起的肝细胞凋亡的调节过程中都是一个中心环节,但是该家族是否参与了WD肝损伤的过程及其具体的调节机制还不是很清楚。目前常用于WD研究的动物模型有多种,如LEC大鼠,TX小鼠以及ATP7B基因敲除小鼠,但各模型与WD的病理生理改变并不完全一致,价格昂贵且不易得到,使其进一步的研究受限。本研究的目的在于建立一种简便、可靠的铜过量负荷动物模型,以进一步研究WD铜沉积导致肝脏损伤的发病机理,为WD的临床治疗提供理论依据。【方法】 1.将大鼠随机分为对照组(A组),铜负荷4周组(B组),铜负荷8周组(C组),铜负荷12周组(D组),所有大鼠均按标准饲养12周,A组正常饮食,B组、C组、D组分别从第9周、第5周和第1周起给予含硫酸铜1g/kg的饲料和含0.185%硫酸铜的水。所有大鼠于12周末取血清及肝组织用于检测。 2.采用原子吸收分光光度计测量各实验组大鼠血清及肝组织的铜含量。采用生化分析仪检测各实验组血清转氨酶ALT水平。 3.采用TUNEL法检测各实验组大鼠肝细胞凋亡现象,并计算其凋亡指数(AI)。 4.采用RT-PCR法检测各实验组大鼠肝组织中Bcl-2和Bax mRNA表达水平,并利用凝胶成像分析系统进行了半定量分析。 5.采用免疫组化方法检测各实验组大鼠病理组织切片中Bcl-2和Bax蛋白的表达水平,并通过图文分析系统进行蛋白表达的半定量分析。【结果】 1.铜过量负荷后,大鼠血清及肝组织中的铜含量逐渐升高,12周达到最高水平(血清铜为16.43±4.51 mg/kg,肝组织铜为4.53±0.44 mg/kg)。 2.铜过量负荷后,大鼠血清ALT水平逐渐升高,12周达到最高水平(200.50±16.15 U/L)。 3.随着铜负荷时间的延长,肝细胞的凋亡逐渐增多,即肝细胞凋亡指数逐步升高,铜负荷12周凋亡指数达到最高水平(AI=19.8±1.2%)。 4.各铜负荷组大鼠与对照组相比,肝组织Bax的mRNA表达水平明显高于对照组,且随着铜负荷时间的延长有升高趋势,Bcl-2的mRNA表达水平高于对照组,随铜负荷时间的延长也有升高趋势,但升高幅度低于Bax,故mRNA水平Bcl-2与Bax的比值(Bcl/Bax)随铜负荷时间延长逐步降低。肝组织Bax和Bcl-2的蛋白表达水平与mRNA表达水平的变化基本相一致,故蛋白水平Bcl-2与Bax的比值(Bcl/Bax)随铜负荷时间延长逐步降低。【结论】 1.本研究成功建立了铜过量负荷大鼠模型,该模型可用于研究铜过量沉积导致肝损伤的发病机理。 2.本研究发现铜过量沉积大鼠肝组织可以诱发大量肝细胞凋亡的发生,同时也检测到随着铜负荷时间的延长,Bax/Bcl-2水平逐步升高,推测肝铜沉积引起肝损伤,其机制可能为通过相对上调促凋亡基因Bax水平来诱导肝细胞的凋亡。
[Abstract]:[background and purpose]
Hepatolenticular degeneration (Wilson disease WD) is a disease of autosomal recessive disorder of copper metabolism, the incidence rate is about 3/10 million.WD good in young people, the cause is due to mutations in the ATP7B gene cause ATP7B function defects, can not remove the body of excess copper, lead to copper deposition in the liver tissue in clinical. With chronic liver disease, the symptoms of kidney damage and nervous system disorders. But the WD copper metabolism originated in the liver, the main parts of abnormal clinical manifestations of patients in the liver, more than 50% of the liver function, pathological changes in the liver caused by excessive copper was mainly necrosis of liver cells, and portal vein peripheral inflammation and fibrosis.
The specific mechanism of liver injury caused by copper deposition in the liver is not clear, the domestic and foreign researches indicate that plays an important role in the apoptosis process of injury, but the adjustment process imbalance in the specific regulatory mechanism is not clear.Bcl-2 protein family ratio induced by various causes apoptosis of liver cells is a center link, but the family is involved in the process of WD liver injury and its specific regulation mechanism is not very clear. There are a variety of animal models are commonly used in WD research, such as the LEC rat, TX mice and ATP7B knockout mice, but the pathological changes of each model and WD is not entirely consistent, expensive it is not easy to find, so further research is limited.
The aim of this study is to establish a simple and reliable animal model of copper overload, so as to further study the pathogenesis of liver injury caused by WD copper deposition, and to provide a theoretical basis for the clinical treatment of WD.
[method]
1. rats were randomly divided into control group (group A), copper loading 4 weeks group (B group), copper loading 8 weeks group (C group), copper loading 12 weeks group (D group), all rats were fed for 12 weeks according to the standard A group, normal diet, group B, group C the D group, respectively from the ninth week, fifth weeks and first weeks treated with copper sulfate 1g/kg feed and containing 0.185% copper sulfate water. All the rats in the 12 week, the serum and liver tissue for testing.
2., the content of copper in serum and liver tissue of each experimental group was measured by atomic absorption spectrophotometer. The level of serum aminotransferase ALT in each experimental group was detected by biochemical analyzer.
3. the apoptosis of rat hepatocytes was detected by TUNEL method and the apoptosis index (AI) was calculated.
4. the expression of Bcl-2 and Bax mRNA in the liver tissues of each experimental group was detected by RT-PCR method, and the semi quantitative analysis was carried out by the gel imaging analysis system.
5. immunohistochemical method was used to detect the expression level of Bcl-2 and Bax protein in pathological sections of rats in each experimental group, and semi quantitative analysis of protein expression was performed by graphic analysis system.
[results]
1. after copper overload, the copper content in serum and liver tissue increased gradually, and reached the highest level in 12 weeks (serum copper was 16.43 + 4.51 mg/kg, liver tissue copper was 4.53 + 0.44 mg/kg).
After 2. copper overload, the serum ALT level of rats increased gradually and reached the highest level at the 12 week (200.50 + 16.15 U/L).
3., with the prolongation of copper loading time, the apoptosis of hepatocytes increased gradually, that is, the apoptotic index of hepatocytes gradually increased, and the apoptotic index reached the highest level at 12 weeks after loading (AI=19.8 + 1.2%).
The 4. copper overload rats compared with control group, the expression level of liver Bax mRNA was significantly higher than the control group, and with the extension of the copper loading time increased, the expression level of Bcl-2 mRNA was higher than the control group, with increasing copper load time is also increased, but the increase amplitude was lower than Bax, so the ratio of mRNA the level of Bcl-2 and Bax (Bcl/Bax) gradually decreased with time prolonged. Copper loading levels and mRNA expression of hepatic Bax and Bcl-2 protein expression were consistent, so the ratio of protein levels of Bcl-2 and Bax (Bcl/Bax) gradually decreased with the copper loading time prolonged.
[Conclusion]
1. this study successfully established a rat model of overdose of copper, which can be used to study the pathogenesis of liver injury caused by overdeposition of copper.
2. this study found that excessive copper deposition in liver tissue of rats can induce a large number of liver cell apoptosis, but also detected with increasing copper loading time, the level of Bax/Bcl-2 increased gradually, that of liver copper deposition induced liver injury, apoptosis and its possible mechanism for apoptosis promoting gene Bax level by up to the relative induction of liver cell.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R-332

【引证文献】