【作者】 李宁；

【导师】 袁晓梅；

【作者基本信息】 新乡医学院 ， 临床医学硕士（专业学位）， 2022， 硕士

【摘要】 背景肺纤维化是一种病因复杂的慢性间质性肺病,其预后较差,且发病机制尚未完全明确。赖氨酸特异性的去甲基化酶2A(lysine-specific demethylase 2A,KDM2A)在维持染色质状态与基因激活中起重要作用,其表达水平异常与多种疾病相关。最近研究发现敲除细胞内KDM2A基因可下调转化生长因子β1(transforming growth factor-β1,TGF-β1)的信号表达,而TGF-β1是肺纤维化发展进程中主要的介导因子,提示KDM2A可能参与调控肺纤维化的形成。但目前对于KDM2A的研究主要集中在肿瘤领域,在肺纤维化中的研究相对较少。本实验旨在构建小鼠肺纤维化模型,探讨KDM2A在肺纤维化进展中的表达变化。目的探讨KDM2A在肺纤维化进展中的表达变化,以期为寻求肺纤维化的发病机制提供具有潜力的新方向。方法1.将8周到10周的C57BL/6J雄性小鼠,适应性饲养1周随机分配为3组,分别为正常组(n=11)、14 d模型组(n=11)与21d模型组(n=11)。取模型组小鼠麻醉后,用泪道冲洗针吸取博来霉素(bleomycin,BLM)工作液(剂量2 mg/kg),将液体缓慢注入小鼠气管内诱导建立肺纤维化模型。分别于造模后第14天、第21天取材14 d模型组与21 d模型组小鼠肺组织,最后取材正常组小鼠肺组织。2.通过HE染色、Masson染色观察各组肺组织病理学改变,并参照Szapiel法进行肺泡炎评分与纤维化程度评分。3.免疫组化分析各组肺组织α平滑肌动蛋白(α-smooth muscle actin,α-SMA)、纤维连接蛋白(Fibronectin,FN)、TGF-β1及KDM2A蛋白表达情况并进行免疫组化评分。4.Western blot检测各组肺组织α-SMA、FN、TGF-β1及KDM2A蛋白表达量。结果1.HE、Masson染色(1)正常组肺结构正常,肺泡内未见明显炎性细胞浸润,肺间质无纤维化改变;(2)14 d模型组肺结构受到不同程度破坏,肺泡间隔普遍增厚,肺泡内大量炎性细胞浸润,各级支气管及肺间质内均可清楚的观察到蓝色胶原纤维沉积;(3)21 d模型组肺结构破坏程度明显,肺泡间隔明显增宽,部分肺泡出现塌陷融合,甚至连接形成纤维板,肺泡内仍有大量炎性细胞浸润,在各级支气管、肺间质及纤维化实变区均可观察到大片蓝色胶原纤维沉积。2.肺泡炎、纤维化程度评分(1)与正常组相比,14 d模型组肺泡炎评分明显增加(P<0.001);21d模型组较14 d模型组相比,肺泡炎评分轻度增加,但差异无统计学意义(P>0.05)。(2)与正常组相比,14 d模型组纤维化评分明显增加(P<0.001);21 d模型组较14 d模型组相比,纤维化评分明显增加(P<0.001)。3.免疫组织化学染色与正常组相比,14 d模型组α-SMA(P<0.001)、FN(P<0.001)、TGF-β1(P<0.001)、KDM2A(P<0.001)蛋白免疫组化评分明显升高;21 d模型组较14 d模型组相比,α-SMA(P<0.001)、FN(P<0.05)、TGF-β1(P<0.001)、KDM2A(P<0.01)蛋白免疫组化评分升高。4.Western blot与正常组相比,14d模型组α-SMA(P<0.001)、FN(P<0.001)、TGF-β1(P<0.001)、KDM2A(P<0.001)蛋白表达量明显升高;21d模型组较14d模型组相比,α-SMA(P<0.001)、FN(P<0.01)、TGF-β1(P<0.001)、KDM2A(P<0.001)蛋白表达量升高。5.相关性分析在小鼠肺组织中TGF-β1与KDM2A的蛋白表达量随着纤维化程度的增加而出现不同程度的上升,对两个蛋白表达结果进行相关性分析发现,TGF-β1与KDM2A蛋白表达呈正相关性,相关系数r=0.9161,P<0.0001。结论KDM2A在肺纤维化进展中表达逐渐增强,并与TGF-β1表达呈正相关性,其可能通过调控TGF-β1的表达来参与肺纤维化的形成。更多还原

【Abstract】 BackgroundPulmonary fibrosis is a chronic interstitial lung disease with complex etiology,with a poor prognosis and an unclear pathogenesis.Lysine-specific demethylase 2A(Lysine-specific demethylase 2A,KDM2A)plays an important role in maintaining chromatin state and gene activation,and its abnormal expression level is associated with many diseases.Recent studies have found that knocking down the KDM2 A gene in cells can down-regulate the signal expression of transforming growth factor-β1(TGF-β1),and TGF-β1 is the main mediator in the development of pulmonary fibrosis,suggesting that KDM2 A may Involved in regulating the formation of pulmonary fibrosis.However,the current research on KDM2 A mainly focuses on the tumor field,and the research on pulmonary fibrosis is relatively rare.The purpose of this experiment was to construct a mouse model of pulmonary fibrosis and to explore the expression changes of KDM2 A in the progression of pulmonary fibrosis.ObjectiveTo explore the expression changes of KDM2 A in the progression of pulmonary fibrosis,in order to provide a potential new direction for seeking the pathogenesis of pulmonary fibrosis.Methods1.C57BL/6J male mice aged 8 to 10 weeks were randomly assigned to 3 groups after adaptive feeding for 1 week,namely control group(n = 11),14 d model group(n =11)and 21 d model group(n = 11).After the mice in the model group were anesthetized,bleomycin(BLM)working solution(dose 2 mg/kg)was drawn with a lacrimal duct irrigation needle,and the liquid was slowly injected into the trachea of the mice to establish a pulmonary fibrosis model.On the 14 th day and the 21 st day after modeling,the lung tissues of mice in the 14 day model group and the 21 day model group were collected,and finally the lung tissues of the control group mice were collected.2.The pathological changes of lung tissue in each group were observed by HE staining and Masson staining,and the scores of alveolitis and fibrosis were performed according to the Szapiel method.3.Immunohistochemical analysis of α-smooth muscle actin(α-SMA),fibronectin(FN),transforming growth factor-β1(TGF-β1)and KDM2 A protein expression and scored by immunohistochemistry.4.Western blot was used to detect the expression of α-SMA,FN,TGF-β1 and KDM2 A protein in lung tissue of each group.Results1.HE,Masson staining(1)In the control group,the lung structure was normal,there was no inflammatory cell infiltration in the alveoli,and there was no fibrotic change in the lung interstitium;(2)In the 14 day model group,the lung structure was damaged to varying degrees,the alveolar septum was generally thickened,a large number of inflammatory cells were infiltrated in the alveoli,and the blue collagen fiber deposition could be clearly observed in the bronchi and pulmonary interstitium at all levels;(3)In the 21 day model group,the damage to the lung structure was obvious,the alveolar septa were significantly widened,and some alveoli collapsed and merged,and even formed fibrous plates.Large areas of blue collagen fiber deposition were observed.2.Scoring of alveolitis and fibrosis(1)Compared with the control group,the alveolitis score of the 14 day model group was significantly increased(P < 0.001);compared with the 14 day model group,the alveolitis score of the 21 day model group was slightly increased,but the difference was not statistically significant(P > 0.05).(2)Compared with the control group,the fibrosis score of the 14 d model group was significantly increased(P < 0.001);the 21 d model group had a significantly higher fibrosis score than the 14 d model group(P < 0.001).3.Immunohistochemical StainingCompared with the control group,the protein immunohistochemical scores ofα-SMA(P < 0.001),FN(P < 0.001),TGF-β1(P < 0.001),and KDM2A(P < 0.001)in the model group were significantly increased on the 14 th day;Compared with the 14 day model group,the protein immunohistochemical scores of α-SMA(P<0.001),FN(P <0.05),TGF-β1(P < 0.001)and KDM2A(P < 0.01)in the model group were also increased.4.Western blotCompared with the control group,the protein expressions of α-SMA(P < 0.001),FN(P < 0.001),TGF-β1(P < 0.001)and KDM2A(P < 0.001)in the 14 day model group were significantly increased;Compared with the 14 d model group,the expressions ofα-SMA(P < 0.001),FN(P < 0.01),TGF-β1(P < 0.001)and KDM2A(P < 0.001)were increased.5.Correlation analysisThe protein expressions of TGF-β1 and KDM2 A in mouse lung tissue increased to varying degrees with the increase of fibrosis.The correlation analysis of the two protein expression results showed that TGF-β1 and KDM2 A protein expression were positively correlated sex,correlation coefficient r = 0.9161,P < 0.0001.ConclusionsThe expression of KDM2 A gradually increased in the progression of pulmonary fibrosis,and was positively correlated with the expression of TGF-β1.It may participate in the formation of pulmonary fibrosis by regulating the expression of TGF-β1.更多还原