本文關鍵詞：TGFB-1抗體治療急性百草枯中毒大鼠肺損傷的實驗研究，由筆耕文化傳播整理發(fā)布。

        背景：百草枯,是一種高效能季胺類除草劑,其除草效果好而且價格便宜,在廣大的農(nóng)業(yè)國家應用廣泛,但對人卻有極高的毒性,且無特效藥,是目前人類急性單體中毒中死亡率最高的除草劑。百草枯中毒能導致肺、肝、腎等多器官功能損害,其中對肺臟損害最重,肺損害是百草枯中毒患者的主要死亡原因[1]。百草枯中毒已經(jīng)成為我國內(nèi)科常見的中毒急危重癥,但是目前百草枯誘發(fā)急性肺損傷的機制尚不明確,所以百草枯中毒發(fā)病機制的基礎研究和臨床研究意義非常。在以往的研究中,學者常將百草枯中毒機制的研究集中在活性氧以及脂質過氧化反應對肺臟的損傷上,近年來隨著細胞因子在肺間質纖維化中作用機制的研究進展,百草枯中毒的機制研究也逐漸把焦點轉移到細胞因子的變化對肺損傷的作用機制上來。目前認為肺組織纖維化是肺組織損傷后修復的調(diào)節(jié)失控所致。在發(fā)病過程中肺巨噬細胞吞噬壞死細胞和組織碎片誘發(fā)炎性反應,分泌TGF-β1。而目前的研究認為TGF-β1是一種具有多種生物學效應的細胞因子,并且是促肺纖維化的關鍵細胞因子。以往研究認為TGF-β1的生物學效應主要是由其下游的Smads蛋白信號通道介導,其中Smad2、3、4是該信號通道中的關鍵蛋白。但是近年來隨著對MAPK信號通道的研究進展,特別是Ras/Raf/MEK/ERK信號通道的作用機制和生物學效應日益明確,ERK/MAPK信號通道在肺纖維化中的作用逐漸得到了重視。而且Smad信號通道和ERK信號通道以及其他信號通道蛋白之間具有交叉的相互作用,構成了復雜的細胞因子網(wǎng)絡。菅向東教授在百草枯中毒致肺纖維化大鼠模型肺組織中細胞因子的動態(tài)變化研究時發(fā)現(xiàn)該細胞因子網(wǎng)絡在百草枯致肺纖維化的發(fā)病機制中具有重要作用,并在此基礎上提出了應用TGF-β1抗體治療急性百草枯中毒的假說,認為TGF-β1抗體會將來能夠成為治療百草枯肺損傷的有效治療方法,但未經(jīng)實驗證實。目的：研究TGF-β1、肺羥脯氨酸和ERK1/2蛋白在百草枯中毒大鼠模型中的動態(tài)變化,觀察TGF-β1抗體對百草枯急性肺損傷是否具有治療效果。方法：采用前期成熟的百草枯動物模型實驗方法建立動物模型。成年健康SPF級雄性Wister大鼠90只,隨機分為染毒組、治療組和對照組三組。制備染毒組：首先準確稱量每只大鼠體重,然后按50mg/kg的劑量在實驗開始時一次性給予百草枯灌胃染毒,染毒前將所需的百草枯原液(體積分數(shù)是20%)用蒸餾水稀釋至1ml；制備治療組：首先采用與染毒組大鼠相同的染毒方法給予大鼠染毒。按0.1mg/kg的劑量在染毒后半小時給染毒大鼠TGF-01抗體(濃度1mg/ml)皮下注射,然后每周給予0.1mg/kg TGF-β1抗體皮下注射一次；制備對照組：僅給予0.9%氯化鈉注射液lml灌胃。在實驗期間嚴密觀察并記錄大鼠的精神狀態(tài),毛發(fā)變化以及行為變化等。染毒組、治療組大鼠分別于染毒后第7天,染毒后第14天,染毒后第21天和染毒后第28天經(jīng)麻醉后處死,對照組在實驗結束后處死。并同時留取右側肺葉的肺泡灌洗液和左側肺組織進行病理學和免疫組織化學等檢測。結果：治療組較染毒組TGF-β1、ERK、P-ERK和羥脯氨酸降低,差異有統(tǒng)計學意義。染毒組較對照組的TGF-β1、 ERK、P-ERK和羥脯氨酸升高,差異有統(tǒng)計學意義。光學顯微鏡觀察,肺組織在早期隨著實驗天數(shù)的增加炎性細胞浸潤程度也逐漸加重,而后期炎性細胞浸潤逐漸減輕代之以肺泡間隔纖維化的逐漸形成。同期的治療組與染毒組大鼠的肺組織比較,炎癥逐漸減輕,纖維化程度減輕。染毒組大鼠在實驗開始出現(xiàn)活動減少和精神萎靡,部分大鼠出現(xiàn)口鼻出血以及呼吸困難等缺氧表現(xiàn),治療組大鼠較同期染毒組反應明顯減輕,用藥5-7后活動較同期染毒組逐漸增多,呼吸平穩(wěn),缺氧癥狀減輕明顯。結論：在百草枯中毒損害的發(fā)病過程中有TGF-β1介導的ERK信號通路參與,TGF-β1在通道中是關鍵因子,TGF-β1抗體在理論上具有治療百草枯中毒肺損害的作用,在本實驗中獲得了良好的治療效果。

    Background:Paraquat is a kind of highly efficient quaternary amine herbicide.Since it’s excellent weed control effect and poor price, paraquat is widely used in ample agriculture country. But for it’s high toxicity to people, and there are no specifics,paraquat poisoning has become the highest humans mortality in acute poisoning by herbicide. Paraquat poisoning can cause lung, liver, kidney and other organ damage, and lung is the most serious damage organ [1]. Paraquat poisoning has become the common severe acute disease in poisoning in the department of internal medicine. The mechanism of the acute lung injury in paraquat poisoning is not clear,and the significance about paraquat poisoning research in basic and clinical is clear. In previous study, Scientists often focused the mechanism on active oxygen and lipid peroxidation in lung injury induced by paraquat poisoning, but the focus has been shifted to the changes of cytokines with the development of pulmonary fibrosis mechanism in recent years.It’s believed the bad repairment is the main mechanism of pulmonary fibrosis. Lung macrophage phagocytose dead cells and pathogens,and inducing inflammation classically,then macrophage induces the TGF-pi. TGF-β1is a multifunctional cytokine,and is the key cell factor of fibrosis.Previous researchs suggest Smads pathway,downstream of TGF-β1,is the primary channel to TGF-β1effection.,and Smad2,Smad3,Smad4have a major role in this channel. With the progress of the MAPK signal channel in recent years, especially in the Ras/Raf/MEK/ERK signal channel mechanism and it’s biological effect, the role of ERK/MAPK signal channels in pulmonary fibrosis gradually got the attention. Smad proteins and ERKproteins and other signal channel has a reciprocal interaction and constitute a complex network.It’s believed that the network play an important role in the process of pulmonary fibrosis. Mr. Jian Xiangdong suggested that TGF-β1antibody will be able to become the more effective treatment about paraquat-induced lung injury, but without the experiment confirmed.Objective:Our research study the therapy of TGF-β1antibody in treating acute lung injury and pulmonary interstitial fibrosis induced by paraquat.Method:All90adult Wister male rats were randomly assigned to three group: paraquat poisoning group, the TGF-β1antibody therapy group and the control group o The paraquat poisoning group(rats were intragastric administration paraquat50mg/kg body weight once at the beginning); the TGF-β1antibody therapy group(rats were given TGF-β1antibody0.lmg/kg Subcutaneous injection30minutes after paraquat was given, then the same dose was given once a week);the control group(rats were intragastric administration with physiological saline). At7th,14th,21st,28th day rats were sacrificed postanesthetic respectively after paraquat exposure, sample of lung tissue and bronchoalveolar lavage fluid were collected. TGF-β1、 ERK1/2and P-ERK of the sample were determined. Optical microscope were performed to examine pathological changes in lung. And observe the experiment animal behavior closely.Results:The dosage of TGF-β1、ERK1/2、P-ERK and hydroxyproline of paraquat poisoning group were significantly higher than the same of the control group, and the paraquat poisoning group was significantly higher than the therapy group in the same time. Under optical microscope, the tissue damage of lung was aggravated, and reduced after TGF-β1antibody was administrated. The paraquat poisoning group rats’ activity appear decreased and spiritual malaise, mouth and nose bleeding,part of rats breathing difficulties because of lack of oxygen, the therapy group rats reaction significantly reduce to the poisoning group rats in the same time, after five to seven days,time poisoning group rats’activity were gradually increased, the oxygen to relieve symptoms release significantly.Conclusion:ERK signaling pathway are involved in pulmonary interstitial fibrosis induced by paraquat poisoning pathogenic processes, TGF-β1is a key factor in the two pathways, In this experiment the TGF-β1antibody has therapeutic effect on acute pulmonary injury,which induced by paraquat poisoning.

TGFB-1抗體治療急性百草枯中毒大鼠肺損傷的實驗研究

目錄4-5CATALOUE5-6中文摘要6-8ABSTRACT8-10符號說明11-12前言12-19材料和方法19-29    1.主要試劑及藥物19    2.動物模型制備19-20    3.標本采集20    4.標本檢測20-28        4.1 酶聯(lián)免疫吸附法(ELISA)檢測TGFβ-120-23        4.2 樣本堿水解法肺組織測定羥脯氨酸23-24        4.3. 免疫組化染色檢測ERK1/2、P-ERK蛋白24-26        4.4. 免疫印跡蛋白定量分析ERK、P-ERK26-28    5.肺部組織學檢查28    6.統(tǒng)計學方法28-29結果29-34    1.實驗動物觀察29    2.肺組織羥脯氨酸含量29    3.肺泡灌洗液、肺組織勻漿中TGF-β1測定結果29-30    4.ERK、P-ERK蛋白免疫組化的結果30-32    5.肺組織大體觀察形態(tài)學變化32-33    6.肺組織病理光鏡檢查(光學顯微觀察)33-34討論34-42結論42-43參考文獻43-54附圖54-61綜述61-67    參考文獻64-67致謝67-68攻讀學位期間發(fā)表的學術論文68-69學位論文評閱及答辯情況表69

  本文關鍵詞：TGFB-1抗體治療急性百草枯中毒大鼠肺損傷的實驗研究，，由筆耕文化傳播整理發(fā)布。