本文選題：博來霉素　切入點(diǎn)：肺纖維化　出處：《北京協(xié)和醫(yī)學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：肺纖維化(pulmonary fibrosis,PF)是以炎癥和細(xì)胞外基質(zhì)沉積為特征,呈進(jìn)展性和致死性的彌漫性肺間質(zhì)疾病;由于疤痕引起肺結(jié)構(gòu)不可逆的破壞,造成通氣障礙,氣體交換受阻,患者因呼吸衰竭而死亡。其中特發(fā)性肺纖維化(Idiopathicpulmonaryfibrosis,IPF)是由未知原因引起的肺纖維化疾病,常見于55-75歲的中老年人,患者確診后的生存期只有2～6年,發(fā)病機(jī)制未闡明,缺乏有效的治療手段。為探究肺纖維化的發(fā)病機(jī)制及評價(jià)新藥的有效性,已建立多種肺纖維化動物模型,如博來霉素(Bleomycin,BLM)、石棉、二氧化硅、輻射等方式誘導(dǎo)嚙齒類動物模型。常用的是小鼠博來霉素單次染毒模型,能一定程度上模擬肺纖維化的一些相關(guān)特點(diǎn),但是發(fā)病呈急性過程,與臨床發(fā)病不同。為此本課題首先比較了BLM多次氣管滴注染毒與單次染毒肺部及全身改變。將C57BL/6雄性小鼠隨機(jī)分為對照組、BLM單次染毒組、BLM多次染毒組,BLM單次染毒組染毒劑量為3.5mg/kg,BLM多次染毒組BLM染毒濃度為0.4mg/ml,每只100ul,2周一次。在染毒2周、4周、8周時(shí)觀察三組小鼠的體重變化、外周血計(jì)數(shù)、臟器指數(shù)(肺、腎臟、胸腺、脾臟)、肺組織HE染色及肺組織羥脯氨酸含量等指標(biāo)。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)BLM單次染毒模型小鼠在2w時(shí)病理評分最高,4w時(shí)評分降低,表明肺局部反應(yīng)減輕,BLM多次染毒模型小鼠肺病理評分相對穩(wěn)定,肺損傷持續(xù)存在;BLM多次染毒小鼠肺組織中的羥脯氨酸含量高于單次染毒小鼠;而兩組小鼠的體重、外周血計(jì)數(shù)和對照組小鼠相比沒有差異,表明肺局部反應(yīng)BLM多次染毒模型優(yōu)于單次染毒模型,兩種染毒方式對小鼠的全身反應(yīng)不明顯。在此基礎(chǔ)上,我們建立小鼠BLM多次染毒模型,觀察肺組織病變變化規(guī)律,將小鼠分為對照組和模型組,模型組BLM染毒濃度為0.4mg/ml,每只100ul,2周一次,染毒8次,觀察肺指數(shù)、肺組織HE染色、Masson染色、肺組織SA β-gal染色等指標(biāo)。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)模型小鼠具有肺纖維化進(jìn)展不可逆,在16w時(shí)有衰老細(xì)胞存在,提示細(xì)胞衰老與肺纖維化有一定關(guān)系。ABT263是BCL-2和BCL-xL的抑制劑,前期研究發(fā)現(xiàn)ABT263對衰老細(xì)胞有選擇性毒性。為探討ABT263對BLM誘導(dǎo)肺纖維化的治療作用,課題的第二部分首先建立BLM誘導(dǎo)的A549細(xì)胞衰老模型,發(fā)現(xiàn)衰老A549細(xì)胞對ABT263的細(xì)胞毒性更為敏感,提示ABT263對衰老細(xì)胞具有選擇性清除作用。在此基礎(chǔ)上開展了體內(nèi)實(shí)驗(yàn),利用BLM多次染毒肺纖維化模型觀察ABT-263對肺纖維化的治療作用及探究其作用機(jī)制。將C57BL/6雄性小鼠分為四組:對照組(ctrl)、ABT給藥組(ABT)、模型組(BLM)、治療組(BLM+ABT)。在模型組小鼠BLM染毒8次后給藥,ABT給藥組和治療組小鼠}f予ABT263,ABT263給藥劑量為50mg/kg·day,連續(xù)給藥5天,停藥兩周。ABT給藥兩個(gè)療程后取材觀察小鼠體重變化、肺指數(shù)、HE染色及評分、Masson染色、肺組織羥脯氨酸含量、外周血計(jì)數(shù)、SA β-gal染色、BCL-2免疫熒光染色等指標(biāo)。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)治療組小鼠肺組織中衰老細(xì)胞減少、肺組織病理評分降低,研究提示ABT-263可能通過抑制Bcl-2清除衰老細(xì)胞對肺纖維化起到治療和緩解作用。綜上,本課題建立了 BLM氣管滴注多次染毒誘導(dǎo)肺纖維化模型;并且觀察到ABT263可能對BLM引起的肺組織病變具有一定的治療作用。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R563;R-332

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