过表达FOXO1的BMSCs对哮喘模型小鼠肺嗜酸性粒细胞浸润和气道重构的抑制作用及其机制  

Inhibitory effect of BMSCs over-expressing FOXO1 on pulmonary eosinophil infiltration and airway remodeling in asthmatic mice and its mechanism

作　　者：何小双[1] 徐丽娜 崔梅 辛雯艳 HE Xiaoshuang;XU Lina;CUI Mei;XIN Wenyan(Department of Respiratory and Critical Care Medicine,First Affiliated Hospital,School of Medical Sciences,Shihezi University,Shihezi 832008,China;Department of Pathology,People’s Hospital,Xinjiang Uygur Autonomous Region,Urumqi 830001,China)

机构地区：[1]石河子大学医学院第一附属医院呼吸与危重症医学科,新疆石河子832008 [2]新疆维吾尔自治区人民医院病理科,新疆乌鲁木齐830001

出　　处：《吉林大学学报（医学版）》2023年第5期1192-1201,共10页Journal of Jilin University：Medicine Edition

基　　金：新疆生产建设兵团科技局科技攻关与成果转化计划项目(2021AD006)。

摘　　要：目的:探讨过表达叉头转录因子1(FOXO1)的骨髓间充质干细胞(BMSCs)对哮喘小鼠肺嗜酸性粒细胞浸润和气道重构的抑制作用,并阐明其可能的作用机制。方法:分离小鼠的BMSCs,采用油红O染色和茜素红染色鉴定BMSCs,流式细胞术鉴定BMSCs的表型。取对数生长期的BMSCs,分为对照组(不进行处理),FOXO1-BMSCs组(感染携带FOXO1基因的重组慢病毒)和NC-BMSCs组(感染阴性对照重组慢病毒)。采用实时荧光定量PCR(RT-qPCR)法检测各组BMSCs中FOXO1 mRNA表达水平,Western blotting法检测各组BMSCs中FOXO1蛋白表达水平。将40只小鼠随机分为对照组(给予生理盐水)、模型组(给予生理盐水)、NC-BMSCs组(给予NCBMSCs悬液)和FOXO1-BMSCs组(给予FOXO1-BMSCs悬液),每组10只。除对照组外,其他3组小鼠采用卵清蛋白(OVA)和雾化激发的方法制备哮喘动物模型。检测各组小鼠BALF中细胞分类计数,HE染色观察各组小鼠肺组织病理形态表现,免疫荧光法检测各组小鼠肺组织中α-平滑肌肌动蛋白(α-SMA)和增殖细胞核蛋白(PCNA)表达情况,Image-Pro Plus软件测量各组小鼠支气管管腔内周长(Pi)、管壁面积(W)、支气管平滑肌面积(S)和支气管平滑肌细胞核数目(N),并计算S/Pi、W/Pi和N/Pi,Western blotting法检测各组小鼠肺组织中基质金属蛋白酶9(MMP-9)、基质金属蛋白酶12(MMP-12)和基质金属蛋白酶组织抑制因子1(TIMP-1)蛋白表达水平。结果:分离培养的BMSCs呈纺锤形,油红O染色和茜素红染色后可见明显红色脂滴形成和橘红色沉淀,BMSCs表面CD29、CD44和CD71表达量升高,CD34、CD45和HLA-DR表达量降低。与对照组和NCBMSCs组比较,FOXO1-BMSCs组BMSCs中FOXO1 mRNA和蛋白表达水平升高(P<0.05)。与模型组和NC-BMSCs组比较,FOXO1-BMSCs组小鼠支气管肺泡灌洗液(BALF)中嗜酸性粒细胞、巨噬细胞、淋巴细胞和中性粒细胞数减少(P<0.05),气道及肺泡中炎性细胞浸润程度减轻,S/Pi、W/Pi和N/Pi降低(P<0.0Objective:To discuss the inhibitory effect of bone marrow-derived mesenchymal stem cells(BMSCs)over-expressing forkhead box transcription factor 1(FOXO1)on the eosinophil infiltration and airway remodeling of the asthmatic mice,and to clarify the possible mechanism.Methods:The BMSCs of the mice were isolated,and Oil Red O staining and Alizarin staining were used to identify the BMSCs.Flow cytometry was used to identify the phenotypes of the BMSCs.The BMSCs at logarithmic growth phase were collected and divided into control group(without treatment),FOXO1-BMSCs group(infected with recombinant lentivirus carrying FOXO1 gene),and NC-BMSCs group(infected with negative control recombinant lentivirus).Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of FOXO1 mRNA in the BMSCs in various groups;Western blotting method was used to detect the expression levels of FOXO1 protein in the BMSCs in various gorups.Forty mice were randomly divided into control group(given saline),model group(given saline),NC-BMSCs group(given NC-BMSCs cell suspension),and FOXO1-BMSCs group(given FOXO1-BMSCs cell suspension),and there were 10 mice in each group.Except for control group,the mice in the other three groups were sensitized with ovalbumin(OVA)and challenged with aerosol to establish the asthma models.The cell classification counts in bronchoalveolar lavage fluid(BALF)of the mice in various groups were detected;the histopathology of lung tissue of the mice in various groups was observed by HE staining;the expressions ofα-smooth muscle actin(α-SMA)and proliferating cell nuclear antigen(PCNA)in lung tissue of the mice in various groups were detected by immunofluorescence method;the bronchial lumen circumference(Pi),wall area(W),smooth muscle area(S),and numbers of smooth muscle cell nuclei(N)of the mice in various groups were detected by using Image-Pro Plus Software,and the ratios of S/Pi,W/Pi,and N/Pi were calculated;the expression levels of matrix metalloproteinase-9(MMP-9),matrix metallo

关 键 词：哮喘 叉头转录因子1 骨髓间充质干细胞 嗜酸性粒细胞 气道重塑 

分 类 号：R-332[医药卫生] R562.25