發(fā)布時間：2018-02-26 09:01

  本文關(guān)鍵詞： WD 肝細胞凋亡 Bax Bcl-2　出處：《華中科技大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 【背景及目的】 肝豆狀核變性(Wilson disease, WD)是常染色體隱性遺傳的銅代謝障礙疾病,發(fā)病率約為3/10萬。WD好發(fā)于青少年,其病因是由于ATP7B基因突變導(dǎo)致ATP7B功能出現(xiàn)缺陷,不能清除體內(nèi)多余的銅,導(dǎo)致銅在肝臟等組織中沉積。臨床上以慢性肝臟病變、腎臟功能損害以及神經(jīng)系統(tǒng)障礙等癥狀為主。但WD的銅代謝障礙起源于肝臟,主要異常部位也在肝臟,50%以上的病人臨床表現(xiàn)為肝功能減退、肝中過多的銅引起的病理改變主要為肝細胞壞死、門靜脈及其周圍炎癥和纖維化。 目前對銅沉積在肝臟引起肝臟損傷的具體機制還不明確,國內(nèi)外的研究表明凋亡在此損傷過程中起著重要的作用,但是其具體的調(diào)節(jié)機制并不明確。Bcl-2家族調(diào)控蛋白比例的失衡在多種原因引起的肝細胞凋亡的調(diào)節(jié)過程中都是一個中心環(huán)節(jié),但是該家族是否參與了WD肝損傷的過程及其具體的調(diào)節(jié)機制還不是很清楚。目前常用于WD研究的動物模型有多種,如LEC大鼠,TX小鼠以及ATP7B基因敲除小鼠,但各模型與WD的病理生理改變并不完全一致,價格昂貴且不易得到,使其進一步的研究受限。 本研究的目的在于建立一種簡便、可靠的銅過量負荷動物模型,以進一步研究WD銅沉積導(dǎo)致肝臟損傷的發(fā)病機理,為WD的臨床治療提供理論依據(jù)。 【方法】 1.將大鼠隨機分為對照組(A組),銅負荷4周組(B組),銅負荷8周組(C組),銅負荷12周組(D組),所有大鼠均按標準飼養(yǎng)12周,A組正常飲食,B組、C組、D組分別從第9周、第5周和第1周起給予含硫酸銅1g/kg的飼料和含0.185%硫酸銅的水。所有大鼠于12周末取血清及肝組織用于檢測。 2.采用原子吸收分光光度計測量各實驗組大鼠血清及肝組織的銅含量。采用生化分析儀檢測各實驗組血清轉(zhuǎn)氨酶ALT水平。 3.采用TUNEL法檢測各實驗組大鼠肝細胞凋亡現(xiàn)象,并計算其凋亡指數(shù)(AI)。 4.采用RT-PCR法檢測各實驗組大鼠肝組織中Bcl-2和Bax mRNA表達水平,并利用凝膠成像分析系統(tǒng)進行了半定量分析。 5.采用免疫組化方法檢測各實驗組大鼠病理組織切片中Bcl-2和Bax蛋白的表達水平,并通過圖文分析系統(tǒng)進行蛋白表達的半定量分析。 【結(jié)果】 1.銅過量負荷后,大鼠血清及肝組織中的銅含量逐漸升高,12周達到最高水平(血清銅為16.43±4.51 mg/kg,肝組織銅為4.53±0.44 mg/kg)。 2.銅過量負荷后,大鼠血清ALT水平逐漸升高,12周達到最高水平(200.50±16.15 U/L)。 3.隨著銅負荷時間的延長,肝細胞的凋亡逐漸增多,即肝細胞凋亡指數(shù)逐步升高,銅負荷12周凋亡指數(shù)達到最高水平(AI=19.8±1.2%)。 4.各銅負荷組大鼠與對照組相比,肝組織Bax的mRNA表達水平明顯高于對照組,且隨著銅負荷時間的延長有升高趨勢,Bcl-2的mRNA表達水平高于對照組,隨銅負荷時間的延長也有升高趨勢,但升高幅度低于Bax,故mRNA水平Bcl-2與Bax的比值(Bcl/Bax)隨銅負荷時間延長逐步降低。肝組織Bax和Bcl-2的蛋白表達水平與mRNA表達水平的變化基本相一致,故蛋白水平Bcl-2與Bax的比值(Bcl/Bax)隨銅負荷時間延長逐步降低。 【結(jié)論】 1.本研究成功建立了銅過量負荷大鼠模型,該模型可用于研究銅過量沉積導(dǎo)致肝損傷的發(fā)病機理。 2.本研究發(fā)現(xiàn)銅過量沉積大鼠肝組織可以誘發(fā)大量肝細胞凋亡的發(fā)生,同時也檢測到隨著銅負荷時間的延長,Bax/Bcl-2水平逐步升高,推測肝銅沉積引起肝損傷,其機制可能為通過相對上調(diào)促凋亡基因Bax水平來誘導(dǎo)肝細胞的凋亡。
[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.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R-332

【引證文獻】

相關(guān)碩士學(xué)位論文 前1條

1 趙漢嵩;亞慢性染鋁致大鼠肝損傷的研究[D];東北農(nóng)業(yè)大學(xué);2011年

，

本文編號：1537351