摘要
目的研究脂肪间充质干细胞(AD-MSC)在利拉鲁肽(Liraglutide)的协同作用下对脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)的保护作用。方法将体外培养的AD-MSC细胞悬液充分混匀后按照相同的细胞数量和体积分为3组:对照组、LPS组(30 mg/L)以及LPS(30 mg/L)+利拉鲁肽(10 nmol/L)组。MTT实验检测6、24、48、72 h时各组AD-MSC的增殖情况;AnnexinV-FITC/PI双染流式细胞术检测3组细胞的凋亡情况;体外培养72 h,Western blot检测3组细胞凋亡相关蛋白cleaved caspase-3、Bax及Bcl-2的表达。体内实验中将60只雄性SPF级BALB/c小鼠,按照随机数字表随机分为5组:对照组、急性肺损伤模型组(ALI组)、ALI+脂肪间充质干细胞组(ALI+AD-MSC组)、ALI+利拉鲁肽组(ALI+Liraglutide组)、ALI+脂肪间充质干细胞+利拉鲁肽组(ALI+AD-MSC+Liraglutide组),观察小鼠的一般情况,分别在LPS刺激造模后2 d和7 d取小鼠肺组织,HE染色检查小鼠肺部的病理变化,并进行肺损伤评分;免疫组织化学检测小鼠肺组织,观察间充质干细胞标志性蛋白Nanog的表达变化;收集小鼠BALF,瑞氏-姬姆萨染色法计数BALF中性粒细胞数量;测定小鼠肺组织湿/干比重(W/D)。结果AD-MSC在LPS的刺激下细胞凋亡明显高于对照组(P<0.05),在6、24、48和72 h的增殖作用明显低于对照组(均P<0.05)。加入利拉鲁肽后改善了AD-MSC的凋亡(P<0.05),促进了细胞在6、24、48和72 h的增殖作用。与对照组相比,在2 d和7 d模型组中,ALI组小鼠肺损伤病理严重,BALF中性粒细胞数量增多[分别为(65.63±1.34和1.74±0.17、51.67±1.35和1.55±0.13)×104/ml(均P<0.05)],小鼠肺组织W/D增大。Nanog蛋白在7 d模型组中表达量少。与ALI组相比,在2 d和7 d模型组中,ALI+AD-MSC组、ALI+Liraglutide组以及ALI+AD-MSC+Liraglutide组小鼠肺损伤病理程度减轻;BALF中性粒细胞数量在ALI+AD-MSC组[(37.04±1.23、29.17±0.68)×104/ml(均P<0.05)]、ALI+Liraglutide组[(39.58±1.67、35.42±0.25)×104/ml(均P<0
Objective To explore the protective effect of human adipose-derived mesenchymal stem cells(AD-MSCs)and liraglutide on lipopolysaccharide(LPS)-induced acute lung injury(ALI).Methods AD-MSCs were cultured in vitro and randomly divided into 3 groups:control group,LPS group(30 mg/L),and LPS(30 mg/L)+liraglutide(10 nM)group.MTT assay was used to detect the proliferation of AD-MSCs at 6,24,48 and 72 h.Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis of the cells.Western blot was used to detect the expression of apoptotic proteins cleaved caspase-3,Bax and Bcl-2 at 72 h in vitro.For the in vivo experiment,60 male SPF BALB/c mice were randomly divided into 5 groups:control group,ALI group,ALI+AD-MSCs group,ALI+Liraglutide group,and ALI+AD-MSCs+Lraglutide group.The mice were sacrificed on day 2 and day 7 after LPS treatment.HE staining was used to examine the pathological changes of the lungs of mice,and the scores of lung injury were measured.The lung tissues of mice were examined by immunohistochemistry,and the expression of the marker protein Nanog of mesenchymal stem cells was observed.BALF was collected,and the number of BALF neutrophils was counted by Rayleigh Giemsa staining.The wet/dry specific gravity of mouse lung tissue was recorded.Results The apoptosis of AD-MSCs stimulated by LPS was significantly higher than that of the control group(P<0.05),and the proliferation of AD-MSCs at 6,24,48 and 72 h was significantly lower than that of the control group(all P<0.05).The addition of Liraglutide reduced the apoptosis of AD-MSCs(P<0.05),and promoted the proliferation of AD-MSC at 6,24,48 and 72 h.Compared with the control group,in the 2 d and 7 d model groups,the lung injury pathology of ALI group had lung injury,increased number of neutrophils in BALF(65.63±1.34 vs 1.74±0.17,51.67±1.35 vs 1.55±0.13)×104/ml(all P<0.05),and increased W/D of lung tissues.The expression level of Nanog protein was low in the 7 d model group.Compared with the ALI group,in 2 d and 7 d model groups,the
作者
冯耘
杨晓彤
王林林
瞿介明
宋元林
Feng Yun;Yang Xiaotong;Wang Linlin;Qu Jieming;Song Yuanlin(Department of Pulmonary and Critical Care Medicine,Zhongshan Hospital,Fudan University,Shanghai 200032,China;School of Pharmacy,East China,University of Science and Technology,Shanghai 200237,China;Department of Pulmonary and Critical Care Medicine,Shanghai Ruijin Hospital,School of Medicine,Shanghai Jiaotong University,Shanghai 200025,China)
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2020年第9期765-771,共7页
Chinese Journal of Tuberculosis and Respiratory Diseases
基金
上海交通大学医学院高水平创新团队激励计划(协同创新团队)。
