【作者】 王敏；

【导师】 夏书月；

【作者基本信息】 沈阳医学院 ， 内科学（专业学位）， 2022， 硕士

【摘要】 目的本研究旨在探索骨髓间充质干细胞源外泌体(Bone marrow Mesenchymal Stem Cells-derived Exosomes,BMSC-Exos)通过雾化吸入对脂多糖(LPS)及香烟诱导的慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,COPD)大鼠上皮-间质转化(Epithelial Mesenchymal Transition,EMT)过程的抑制作用,并探讨其作用机制。方法1.骨髓间充质干细胞的分离、鉴定及外泌体的提取采用全骨髓贴壁法培养大鼠BMSCs,并用流式细胞术进行检测。应用外泌体浓缩试剂盒及纯化试剂盒分离纯化第三代BMSCs来源的外泌体,并通过透射电子显微镜(Transmission Electron Microscope,TEM)、纳米粒子跟踪分析(Nanoparticle Tracking Analysis,NTA)和蛋白质免疫印迹法(Western Blotting)对其进行鉴定。2.COPD大鼠模型的建立应用脂多糖联合熏烟方法建立COPD大鼠模型。实验第1天和第14天,除对照组外每只大鼠气管内注入LPS各200μg/200μl。第2~13、15~28天每日上、下午置于烟熏箱内被动吸烟(焦油25 mg;CO 13 mg;尼古丁1.1 mg)处理,每次0.5h,2次烟熏间隔时间大于或等于6h。对照组大鼠气管内注射等量生理盐水。3.实验分组42只4周龄SD大鼠随机分为7组,每组6只。对照组(Control):气管内滴注和雾化等量的PBS;模型组(Model):LPS滴注联合熏烟造模,雾化等量的PBS;低剂量BMSC-Exos雾化组(Low-MSC-Exos(IN)):造模大鼠,雾化吸入0.5×108 particles/kg的BMSC-Exos悬液1ml;中剂量BMSC-Exos雾化组(Mid-MSC-Exos(IN)):造模大鼠,雾化吸入1.0×108 particles/kg的BMSC-Exos悬液1ml;高剂量BMSC-Exos雾化组(High-MSC-Exos(IN)):造模大鼠,雾化吸入1.5×108particles/kg的BMSC-Exos悬液1ml;BMSC-Exos尾静脉注射组(MSC-Exos(IV)):造模大鼠,尾静脉注射1.5×108particles/kg的BMSC-Exos悬液1ml;激素雾化组(Hormone(IN)):造模大鼠,雾化吸入1ml普米克令舒(1mg/只)。各治疗组连续治疗5天,每次30min。4.雾化BMSC-Exos对COPD大鼠肺组织全身和局部炎症损伤及EMT相关机制的影响在大鼠干预治疗后的第28天,进行肺功能检测,解剖大鼠,收集肺组织样本、血清及BALF。比较各组肺功能情况并且验证COPD模型建立是否成功。采用ELISA检测各组支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)及血清中促纤维化因子β1(TGF-β1)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)水平。使用苏木精和伊红(HE)以及马松染色来评估肺组织的变化。Western blotting检测EMT过程相关标志物的表达,包括E-钙黏蛋白(E-cadherin,E-cad)、细胞角蛋白19(Cytokeratin-19,CK19)和α-平滑肌动蛋白(α-Smooth muscle actin,α-SMA),免疫组织化学染色(IHS)检测Wnt/β-catenin信号通路的标志物的表达,包括p-GSK-3β、GSK-3β、β-catenin和cyclin D1。结果1.BMSCs及其BMSC-Exos的分离和鉴定大鼠BMSCs培养至第3代时呈形状均一的长梭形,成聚集性生长;流式细胞术结果显示第3代BMSCs高表达CD73、CD90和CD105标志物,低表达CD14、CD19、CD34、CD45和HLA-DR标志物;应用分子排阻色谱(SEC)纯化与亲和色谱浓缩相结合的原理分离纯化得到外泌体,TEM观察到提取的外泌体为类椭圆形的囊泡结构,呈典型的杯口状;NTA结果提示BMSC-Exos的峰直径为91.7nm,占比为97.3%,颗粒浓度为3.3×106/ml;Western blotting结果显示外泌体表面蛋白CD9和CD63呈高表达状态。2.各组大鼠肺功能情况与对照组相比,模型组肺功能出现严重损伤,但BMSC-Exos雾化治疗后显著改善了COPD大鼠的肺功能。与模型组相比,雾化BMSC-Exos治疗后,COPD大鼠0.2秒用力呼气容积(FEV0.2)、0.3秒用力呼气容积(FEV0.3)、0.2秒用力呼气容积/用力肺活量(FEV0.2/FVC)、0.3秒用力呼气容积/用力肺活量(FEV0.3/FVC)、呼出25%FVC时呼气流速(FEF25%)和呼出50%FVC时呼气流速(FEF50%)均显著性增加(P<0.05)。3.各组大鼠血清及BALF中TNF-α、IL-6和TGF-β1的水平雾化形式的BMSC-Exos可减轻COPD炎症反应及纤维化进程,其中以低剂量雾化作用最为显著。与模型组相比,BMSC-Exos雾化组、BMSC-Exos尾静脉组及激素组大鼠血清和BALF中TNF-α、IL-6及TGF-β1含量均降低(P<0.05)。其中低剂量BMSC-Exos雾化组的炎症因子及纤维化因子水平较其他治疗组降低的更为显著(P<0.05)。4.各组大鼠肺组织病理学变化在使用BMSC-Exos治疗后显著减少了COPD气道炎症和肺泡结构的破坏。但尾静脉注射BMSC-Exos及雾化激素的治疗效果并不显著。HE染色和masson染色结果显示,对照组大鼠肺泡结构正常,未见明显的炎症细胞的浸润,气管下胶原沉积较少。而模型组大鼠肺泡结构损伤,血管及支气管周围出现大量炎性细胞浸润,蓝色的胶原纤维阳性区域较多。然而经BMSC-Exos雾化治疗后,肺泡融合及炎性细胞浸润减少,蓝色的胶原纤维面积较小,且炎症评分显著降低(P<0.05)。5.不同治疗组EMT相关蛋白水平的表达LPS联合香烟烟雾诱导的COPD,促进了气道上皮细胞的EMT过程,而雾化形式的低、中剂量BMSC-Exos抑制了这一过程。Western blotting结果显示,与对照组相比,模型组上皮细胞标志蛋白CK19和E-cad的表达水平显著减少,间质细胞标志蛋白α-SMA表达水平显著增加(P<0.05)。在低剂量和中剂量BMSC-Exos及激素雾化治疗后,CK19和E-cad表达增加,α-SMA表达减少(P<0.05)。而高剂量BMSC-Exos雾化组和尾静脉组α-SMA的表达水平显著高于模型组(P<0.05)。6.不同治疗组Wnt/β-Catenin信号通路相关蛋白的表达水平BMSC-Exos雾化治疗后Wnt/β-catenin信号通路呈现负调控基因的高表达及下游基因的低表达。免疫组织化学结果显示,与对照组相比,模型组中Wnt/β-Catenin信号通路的下游基因Cyclin D1、β-catenin和P-GSK-3β表达增加,负调控基因GSK-3β表达减少。在低剂量BMSC-Exos雾化后,Cyclin D1、β-catenin和P-GSK-3β的表达减少,GSK-3β的表达增加。而在高剂量BMSC-Exos雾化组、尾静脉组、激素组中β-catenin和P-GSK-3β的表达与模型组相比无明显统计学差异(P<0.05)。结论1.雾化BMSC-Exos可以改善COPD模型大鼠的肺功能、炎症及纤维化损伤,减少上皮细胞转化,抑制EMT过程。2.在COPD模型大鼠中与EMT密切相关的Wnt/β-catenin信号通路异常活化,而雾化BMSC-Exos后衰减了这一信号,这可能是其抑制EMT过程的机制之一。更多还原

【Abstract】 ObjectiveThe aim of this study was to investigate the inhibitory effect of exosomes derived from bone marrow mesenchymal stem cells(BMSC-Exos)on epithelial-mesenchymal transformation(EMT)induced by lipopolysaccharide and cigarette in chronic obstructive pulmonary disease(COPD)rats,and to explore the mechanism of this inhibitory effect.Methods1.Isolation and identification of bone marrow mesenchymal stem cells and extraction of exosomesThe BMSCs were cultured by whole bone marrow adherence method and detected by flow cytometry.The exosomes from the third generation BMSCs were isolated and purified by exosomal concentration kit and purification kit,and identified by transmission electron microscopy(TEM),Nanoparticle Tracking Analysis(NTA)and Western Blotting.2.Establishment of COPD rat modelThe COPD rat model was established by LPS and fumigation.On the 1st and 14 th day,LPS was injected into the trachea of each rat except the control group,200μg/200μl.Passive smoking(tar 25mg;CO 13mg;nicotine 1.1mg)shall be put in the smoke box in the morning and afternoon for 2 ~ 13 and 15 ~ 28 days,and the interval between two fumigations shall be 0.5h each time or equal to 6h.Rats in control group were injected with the same amount of saline.3.Experimental groupsForty-two 4-week-old SD rats were randomly divided into 7 groups,6 in each group.Control group(Control): intratracheal infusion and atomization equivalent amount of PBS;Model group(Model): LPS infusion combined with fumigation model,atomization equivalent amount of PBS;Low dose BMSC-Exos atomization group(Low-MSC-Exos(IN)): model rats,atomization inhalation 0.5×108 particles/kg of BMSC-Exos suspension 1ml;Medium dose BMSC-Exos atomization group(Mid-MSC-Exos(IN)): model rats,atomization inhalation 1.0×108 particles/kg of BMSC-Exos suspension 1ml;High dose BMSC-Exos atomization group(High-MSC-Exos(IN)): model rats,atomization inhalation 1.5×108particles/kg of BMSC-Exos suspension 1ml;BMSC-Exos tail vein injection group(MSC-Exos(IV)):model rats were injected with 1.5×108 particles/kg BMSC-Exos suspension in tail vein;Hormone nebulization group(Hormone(IN)): model rats,atomization inhalation pulmicort(1mg/rat).Each treatment group was treated continuously for 5 days for 30 minutes.4.Effects of atomized BMSC-Ex OS on systemic and local inflammation and EMT-related mechanisms in COPD ratsOn the 28 th day after the intervention treatment,the pulmonary function was measured,the rats were dissected,lung tissue samples,serum and BALF were collected.The pulmonary function of each group was compared and COPD model was established successfully.The levels of pro-fibrosis factor-β1(TGF-β1),interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in BALF and serum were determined by ELISA.Pulmonary tissue changes were assessed using hematoxylin and eosin(HE)and masson staining.Western blotting detects the expression of markers associated with the EMT process,including E-cadherin(E-cad),cytokeratin-19(CK19)andα-smooth muscle actin(α-SMA),and immunohistochemical staining(IHS)detects the expression of markers associated with the Wnt/β-catenin signaling pathway,including P-GSK-3β,GSK-3β,β-catenin and cyclin D1.Results1.Characterization of BMSCs and BMSC-ExosIn the third generation,rat BMSCs showed long spindle shape and aggregated growth.Flow cytometry analysis showed that BMSCs highly expressed of CD73,CD90 and CD105,and lowly expressed of CD14,CD19,CD34,CD45 and HLA-DR.Exosomes were separated and purified by molecular exclusion chromatography(SEC)and affinity chromatography concentration.The results of TEM showed that the exosomes were typical cup-shaped.NTA results indicate that the peak diameter of BMSC-Exos is 91.7nm,accounting for 97.3% of all particles,and the particle concentration was 3.3×106/ml.The results of Western blotting showed that exosome surface proteins CD9 and CD63 were highly expressed.2.Lung function of rats in each groupCompared with the control group,the lung function of the model group was severely damaged,but the lung function of COPD rats was significantly improved after BMSC-Exos atomization.Compared with the model group,the levels of 0.2 second forced expiratory volume(FEV0.2),0.3 second forced expiratory volume(FEV0.3),0.2sec forced expiratory volume/Forced lung capacity(FEV0.2/FVC),0.3sec forced expiratory volume/Forced lung activity(FEV0.3/FVC),Expiratory flow rate at25%FVC(FEF25%),Expiratory flow rate at 50%FVC(FEF50%)were significantly increased in all of the BMSC-Exos atomization groups(P < 0.05)。3.Levels of TNF-α,IL-6 and TGF-β1 in serum and BALF of rats in each groupAtomized exosomes can reduce the inflammatory response and fibrosis process of COPD,and the effect of low dose atomization is the most significant.Compared with model group,the contents of TNF-α,IL-6 and TGF-β1 in serum and BALF were decreased of BMSC-Exos atomization group,BMSC-Exos tail vein group and hormone group(P < 0.05).Among them,the inflammatory factors and fibrosis factors in the low-dose BMSC-Exos atomization group were decreased more significantly than those in the other treatment groups(P < 0.05).4.Pathological changes of lung tissues in each groupAfter treatment with BMSC-Exos,there was a significant reduction in airway inflammation and alveolar destruction in COPD.But the effect of caudal vein injection of BMSC-Exos and aerosol hormone was not significant.HE staining and Masson staining showed that the alveolar structure of the control group was normal,no obvious infiltration of inflammatory cells was observed,and collagen deposition was less under the trachea.Compared with the control group,the alveolar structure of model group was damaged,and a large number of inflammatory cells infiltrated around blood vessels and bronchi,with more blue collagen fiber positive areas.However,after BMSC-Exos atomization treatment,alveolar fusion and inflammatory cell infiltration decreased,the area of blue collagen fibers decreased,and the inflammatory score decreased significantly(P < 0.05).5.Expression of EMT-related proteins in different treatment groupsLPS in combination with cigarette smog-induced COPD promoted EMT processes in airway epithelial cells,whereas low and medium dose BMSC-Exos in atomized form inhibited this process.Western blotting results showed that,compared with control group,The expression levels of CK19 and E-cad in the epithelial cells of the model group were significantly reduced,while α-SMA protein expression level was significantly increased(P < 0.05).The levels of CK19 and E-cad increased and α-SMA decreased after low and medium doses of BMSC-Exos and hormone nebulization treatment(P < 0.05).The level of α-SMA in high dose BMSC-Exos atomization group and tail vein group was significantly higher than that in model group(P < 0.05).6.Expression of Wnt/β-catenin signaling pathway related proteins in different treatment groupsAfter BMSC-Exos atomization treatment,the Wnt/β-catenin signaling pathway showed high expression of negative regulatory genes and low expression of downstream genes.The results of Immunohistochemical showed that the expression of Cyclin D1,β-catenin and P-GSK-3β were increased and GSK-3β expression was decreased in the model group compared with the control group(P < 0.05).The expression of Cyclin D1,β-catenin and P-GSK-3β decreased and GSK-3β increased after low dose of BMSC-Exos atomization(P < 0.05).In the high dose BMSC-Exos atomization group,tail vein group and hormone group,there was no significant difference in the expression of β-catenin and P-GSK-3β compared with the model group(P < 0.05).Conclusion1.Atomized BMSC-Exos can improve lung function,inflammation and fibrosis,reduce epithelial cell transformation and reverse the process of EMT in COPD model rats.2.In COPD model rats,the Wnt/β-catenin signaling pathway,which is closely related to EMT,is abnormally activated,and this signal is attenuated after atomization of BMSC-Exos,which may be one of the mechanisms of its inhibition of EMT process.更多还原