小鼠博来霉素多次染毒肺纤维化模型建立及ABT263治疗作用观察

发布时间：2018-03-20 19:57

本文选题：博来霉素　切入点：肺纤维化　出处：《北京协和医学院》2017年硕士论文　论文类型：学位论文

【摘要】：肺纤维化(pulmonary fibrosis,PF)是以炎症和细胞外基质沉积为特征,呈进展性和致死性的弥漫性肺间质疾病;由于疤痕引起肺结构不可逆的破坏,造成通气障碍,气体交换受阻,患者因呼吸衰竭而死亡。其中特发性肺纤维化(Idiopathicpulmonaryfibrosis,IPF)是由未知原因引起的肺纤维化疾病,常见于55-75岁的中老年人,患者确诊后的生存期只有2～6年,发病机制未阐明,缺乏有效的治疗手段。为探究肺纤维化的发病机制及评价新药的有效性,已建立多种肺纤维化动物模型,如博来霉素(Bleomycin,BLM)、石棉、二氧化硅、辐射等方式诱导啮齿类动物模型。常用的是小鼠博来霉素单次染毒模型,能一定程度上模拟肺纤维化的一些相关特点,但是发病呈急性过程,与临床发病不同。为此本课题首先比较了BLM多次气管滴注染毒与单次染毒肺部及全身改变。将C57BL/6雄性小鼠随机分为对照组、BLM单次染毒组、BLM多次染毒组,BLM单次染毒组染毒剂量为3.5mg/kg,BLM多次染毒组BLM染毒浓度为0.4mg/ml,每只100ul,2周一次。在染毒2周、4周、8周时观察三组小鼠的体重变化、外周血计数、脏器指数(肺、肾脏、胸腺、脾脏)、肺组织HE染色及肺组织羟脯氨酸含量等指标。实验结果发现BLM单次染毒模型小鼠在2w时病理评分最高,4w时评分降低,表明肺局部反应减轻,BLM多次染毒模型小鼠肺病理评分相对稳定,肺损伤持续存在;BLM多次染毒小鼠肺组织中的羟脯氨酸含量高于单次染毒小鼠;而两组小鼠的体重、外周血计数和对照组小鼠相比没有差异,表明肺局部反应BLM多次染毒模型优于单次染毒模型,两种染毒方式对小鼠的全身反应不明显。在此基础上,我们建立小鼠BLM多次染毒模型,观察肺组织病变变化规律,将小鼠分为对照组和模型组,模型组BLM染毒浓度为0.4mg/ml,每只100ul,2周一次,染毒8次,观察肺指数、肺组织HE染色、Masson染色、肺组织SA β-gal染色等指标。实验结果发现模型小鼠具有肺纤维化进展不可逆,在16w时有衰老细胞存在,提示细胞衰老与肺纤维化有一定关系。ABT263是BCL-2和BCL-xL的抑制剂,前期研究发现ABT263对衰老细胞有选择性毒性。为探讨ABT263对BLM诱导肺纤维化的治疗作用,课题的第二部分首先建立BLM诱导的A549细胞衰老模型,发现衰老A549细胞对ABT263的细胞毒性更为敏感,提示ABT263对衰老细胞具有选择性清除作用。在此基础上开展了体内实验,利用BLM多次染毒肺纤维化模型观察ABT-263对肺纤维化的治疗作用及探究其作用机制。将C57BL/6雄性小鼠分为四组:对照组(ctrl)、ABT给药组(ABT)、模型组(BLM)、治疗组(BLM+ABT)。在模型组小鼠BLM染毒8次后给药,ABT给药组和治疗组小鼠}f予ABT263,ABT263给药剂量为50mg/kg·day,连续给药5天,停药两周。ABT给药两个疗程后取材观察小鼠体重变化、肺指数、HE染色及评分、Masson染色、肺组织羟脯氨酸含量、外周血计数、SA β-gal染色、BCL-2免疫荧光染色等指标。实验结果发现治疗组小鼠肺组织中衰老细胞减少、肺组织病理评分降低,研究提示ABT-263可能通过抑制Bcl-2清除衰老细胞对肺纤维化起到治疗和缓解作用。综上,本课题建立了 BLM气管滴注多次染毒诱导肺纤维化模型;并且观察到ABT263可能对BLM引起的肺组织病变具有一定的治疗作用。
[Abstract]:Pulmonary fibrosis (pulmonary fibrosis, PF) is characterized by inflammation and extracellular matrix deposition, a progressive and fatal diffuse interstitial lung disease; lung structure caused by scar due to irreversible damage, caused by ventilation, gas exchange is blocked, due to respiratory failure and death in patients with idiopathic pulmonary fibrosis. (Idiopathicpulmonaryfibrosis, IPF) is caused by unknown reason pulmonary fibrosis disease, common in 55-75 years of age in the elderly patients, the survival period after only 2~6 years, did not clarify the pathogenesis, the lack of effective treatment. Effectiveness of the pathogenesis of pulmonary fibrosis and explore the evaluation of new drugs, has established a variety of pulmonary fibrosis animal models, such as bleomycin (Bleomycin, BLM), asbestos, silica, rodent animal model induced by radiation. The commonly used mouse bleomycin single exposure model, to a certain extent. Some related characteristics of pulmonary fibrosis, but the incidence of acute process, and clinical disease. This paper firstly compares the BLM repeated intratracheal instillation and single exposure pulmonary and systemic change. Male C57BL/6 mice were randomly divided into control group, BLM single exposure group, repeated BLM exposure group, BLM single exposure dose was 3.5mg/kg, BLM repeatedly exposed groups treated with BLM concentration of 0.4mg/ml, each 100ul, once every 2 weeks. In 2 weeks, after 4 weeks, the changes of body weight in three groups were observed at 8 weeks, peripheral blood count, organ index (lung, kidney, thymus, spleen, lung index) tissue HE staining and hydroxyproline in lung tissues. The experimental results showed that BLM single exposure of mice in 2W pathological score highest, 4W score decreased, that reduce the lung local reaction, BLM repeatedly exposed mice lung pathological score of lung injury is relatively stable, persistent BLM times; The content of hydroxyproline in lung tissue of mice exposed to the higher than single exposure mice; and the two group of mice weight, peripheral blood count and control mice showed no difference, showed that pulmonary local reaction BLM multiple exposure model is better than that of the single exposure model, two kinds of treatments on mice body reaction is not obvious. On the basis of this. We established BLM mice model to observe the changes of multiple exposure, lesions of lung tissue, the mice were divided into control group and model group, model group, BLM exposure concentration is 0.4mg/ml, each 100ul, every 2 weeks, after 8 times, observe the lung index, lung tissue HE staining, Masson staining, SA staining and other indicators of beta -gal lung tissue. Experimental results show that the model mice with pulmonary fibrosis progression is irreversible, senescent cells in the presence of 16W, suggesting that cell senescence and pulmonary fibrosis there is a relationship between.ABT263 inhibitor BCL-2 and BCL-xL, previous study found that ABT263 Selective toxicity of senescent cells. To explore the therapeutic effect of ABT263 on BLM induced pulmonary fibrosis, the second part of the thesis firstly established A549 cell aging model induced by BLM, found that the cytotoxicity of A549 cells to ABT263 aging is more sensitive, suggesting that ABT263 has the function of eliminating selectivity of senescent cells. On the basis of in vivo experiment. The treatment effect by BLM multiple exposure pulmonary fibrosis model to observe the effect of ABT-263 on pulmonary fibrosis and explore its mechanism. C57BL/6 male mice were divided into four groups: control group (CTRL), ABT group (ABT), model group (BLM), treatment group (BLM+ABT). In the model group were exposed to BLM 8 after administration, ABT administration group and treatment group were treated with ABT263}f, ABT263 dosage was 50mg/kg day, administered continuously for 5 days, two weeks of.ABT administration after two courses were observed the changes of body weight, lung index, HE staining and M score. Asson staining, the content of hydroxyproline in lung tissue, peripheral blood counts, SA beta -gal staining, BCL-2 staining and other indicators. The experimental results found that senescent cells of mice treated in lung tissue decreased, lung pathological score decreased, it is suggested that ABT-263 may inhibit Bcl-2 removal of senescent cells on pulmonary fibrosis to treatment and relief in summary, this research establishes BLM tracheal instillation of multiple exposure induced pulmonary fibrosis model; and observed ABT263 on BLM induced pulmonary lesions have a certain therapeutic effect.

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R563;R-332

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