TGFB-1抗体治疗急性百草枯中毒大鼠肺损伤的实验研究

  本文关键词：TGFB-1抗体治疗急性百草枯中毒大鼠肺损伤的实验研究，由笔耕文化传播整理发布。

        背景：百草枯,是一种高效能季胺类除草剂,其除草效果好而且价格便宜,在广大的农业国家应用广泛,但对人却有极高的毒性,且无特效药,是目前人类急性单体中毒中死亡率最高的除草剂。百草枯中毒能导致肺、肝、肾等多器官功能损害,其中对肺脏损害最重,肺损害是百草枯中毒患者的主要死亡原因[1]。百草枯中毒已经成为我国内科常见的中毒急危重症,但是目前百草枯诱发急性肺损伤的机制尚不明确,所以百草枯中毒发病机制的基础研究和临床研究意义非常。在以往的研究中,学者常将百草枯中毒机制的研究集中在活性氧以及脂质过氧化反应对肺脏的损伤上,近年来随着细胞因子在肺间质纤维化中作用机制的研究进展,百草枯中毒的机制研究也逐渐把焦点转移到细胞因子的变化对肺损伤的作用机制上来。目前认为肺组织纤维化是肺组织损伤后修复的调节失控所致。在发病过程中肺巨噬细胞吞噬坏死细胞和组织碎片诱发炎性反应,分泌TGF-β1。而目前的研究认为TGF-β1是一种具有多种生物学效应的细胞因子,并且是促肺纤维化的关键细胞因子。以往研究认为TGF-β1的生物学效应主要是由其下游的Smads蛋白信号通道介导,其中Smad2、3、4是该信号通道中的关键蛋白。但是近年来随着对MAPK信号通道的研究进展,特别是Ras/Raf/MEK/ERK信号通道的作用机制和生物学效应日益明确,ERK/MAPK信号通道在肺纤维化中的作用逐渐得到了重视。而且Smad信号通道和ERK信号通道以及其他信号通道蛋白之间具有交叉的相互作用,构成了复杂的细胞因子网络。菅向东教授在百草枯中毒致肺纤维化大鼠模型肺组织中细胞因子的动态变化研究时发现该细胞因子网络在百草枯致肺纤维化的发病机制中具有重要作用,并在此基础上提出了应用TGF-β1抗体治疗急性百草枯中毒的假说,认为TGF-β1抗体会将来能够成为治疗百草枯肺损伤的有效治疗方法,但未经实验证实。目的：研究TGF-β1、肺羟脯氨酸和ERK1/2蛋白在百草枯中毒大鼠模型中的动态变化,观察TGF-β1抗体对百草枯急性肺损伤是否具有治疗效果。方法：采用前期成熟的百草枯动物模型实验方法建立动物模型。成年健康SPF级雄性Wister大鼠90只,随机分为染毒组、治疗组和对照组三组。制备染毒组：首先准确称量每只大鼠体重,然后按50mg/kg的剂量在实验开始时一次性给予百草枯灌胃染毒,染毒前将所需的百草枯原液(体积分数是20%)用蒸馏水稀释至1ml；制备治疗组：首先采用与染毒组大鼠相同的染毒方法给予大鼠染毒。按0.1mg/kg的剂量在染毒后半小时给染毒大鼠TGF-01抗体(浓度1mg/ml)皮下注射,然后每周给予0.1mg/kg TGF-β1抗体皮下注射一次；制备对照组：仅给予0.9%氯化钠注射液lml灌胃。在实验期间严密观察并记录大鼠的精神状态,毛发变化以及行为变化等。染毒组、治疗组大鼠分别于染毒后第7天,染毒后第14天,染毒后第21天和染毒后第28天经麻醉后处死,对照组在实验结束后处死。并同时留取右侧肺叶的肺泡灌洗液和左侧肺组织进行病理学和免疫组织化学等检测。结果：治疗组较染毒组TGF-β1、ERK、P-ERK和羟脯氨酸降低,差异有统计学意义。染毒组较对照组的TGF-β1、 ERK、P-ERK和羟脯氨酸升高,差异有统计学意义。光学显微镜观察,肺组织在早期随着实验天数的增加炎性细胞浸润程度也逐渐加重,而后期炎性细胞浸润逐渐减轻代之以肺泡间隔纤维化的逐渐形成。同期的治疗组与染毒组大鼠的肺组织比较,炎症逐渐减轻,纤维化程度减轻。染毒组大鼠在实验开始出现活动减少和精神萎靡,部分大鼠出现口鼻出血以及呼吸困难等缺氧表现,治疗组大鼠较同期染毒组反应明显减轻,用药5-7后活动较同期染毒组逐渐增多,呼吸平稳,缺氧症状减轻明显。结论：在百草枯中毒损害的发病过程中有TGF-β1介导的ERK信号通路参与,TGF-β1在通道中是关键因子,TGF-β1抗体在理论上具有治疗百草枯中毒肺损害的作用,在本实验中获得了良好的治疗效果。

    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抗体治疗急性百草枯中毒大鼠肺损伤的实验研究

目录4-5CATALOUE5-6中文摘要6-8ABSTRACT8-10符号说明11-12前言12-19材料和方法19-29    1.主要试剂及药物19    2.动物模型制备19-20    3.标本采集20    4.标本检测20-28        4.1 酶联免疫吸附法(ELISA)检测TGFβ-120-23        4.2 样本碱水解法肺组织测定羟脯氨酸23-24        4.3. 免疫组化染色检测ERK1/2、P-ERK蛋白24-26        4.4. 免疫印迹蛋白定量分析ERK、P-ERK26-28    5.肺部组织学检查28    6.统计学方法28-29结果29-34    1.实验动物观察29    2.肺组织羟脯氨酸含量29    3.肺泡灌洗液、肺组织匀浆中TGF-β1测定结果29-30    4.ERK、P-ERK蛋白免疫组化的结果30-32    5.肺组织大体观察形态学变化32-33    6.肺组织病理光镜检查(光学显微观察)33-34讨论34-42结论42-43参考文献43-54附图54-61综述61-67    参考文献64-67致谢67-68攻读学位期间发表的学术论文68-69学位论文评阅及答辩情况表69

  本文关键词：TGFB-1抗体治疗急性百草枯中毒大鼠肺损伤的实验研究，，由笔耕文化传播整理发布。