人骨髓间充质干细胞对小鼠急性肺损伤模型的影响  

Effects of human bone marrow mesenchymal stem cells on acute lung injury model in mice

作　　者：宋娟 冯耘[2] 童琳[1] 毕晶[1] 冯娜娜 周建[1] 宋元林[1] 陈翠翠[1] 王林林[1] Song Juan;Feng Yun;Tong Lin;Bi Jing;Feng Nana;Zhou Jian;Song Yuanlin;Chen Cuicui;Wang Linlin(Department of Respiratory and Critical Care Medicine,Zhongshan Hospital Affiliated to Fudan University,Shanghai 200032,China;Department of Respiratory and Critical Care Medicine,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Shanghai Eighth People′s Hospital,Jiangsu University,Shanghai 200235,China)

机构地区：[1]复旦大学附属中山医院呼吸与危重症医学科,上海200032 [2]上海交通大学医学院附属瑞金医院呼吸与危重症医学科,200025 [3]江苏大学附属上海市第八人民医院,200235

出　　处：《国际呼吸杂志》2021年第23期1767-1773,共7页International Journal of Respiration

基　　金：国家自然科学基金青年科学基金项目(82000087);上海市卫生健康委员会科研课题(20204Y0082);上海市徐汇区科委项目(SHXH201838)。

摘　　要：目的研究人骨髓来源间充质干细胞(MSC)对脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)模型的保护作用。方法实验性研究。24只雄性SPF级BALB/c小鼠,随机分成3组:对照组、ALI组和干预组。在气管滴注LPS(5 mg/kg)造模后2 d取小鼠肺组织,HE染色观察肺损伤的病理改变,并进行肺损伤评分;收集小鼠肺泡灌洗液(BALF),瑞氏-吉姆萨染色法计数白细胞和分类,测定总蛋白和炎症细胞因子白细胞介素1β(IL-1β),白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)浓度;测定肺组织湿干比(W/D);RT-PCR检测半胱天冬酶3(Caspase 3)、角质细胞生长因子2(KGF-2)、表面活性蛋白C(SPC)和增殖细胞核抗原(PCNA)mRNA表达情况;TUNNEL方法检测肺内细胞凋亡;免疫组化检测肺内PCNA;Masson方法检测肺内胶原纤维沉积;小动物活体成像示踪MSC在肺内的定植情况。结果与对照组相比,由LPS诱导的急性肺损伤小鼠模型中肺损伤明显严重(11.75±1.71)分比(2.75±0.96)分,(P<0.01),W/D明显升高(5.23±0.15)比(4.36±0.29),P<0.01,总蛋白水平升高(1181.80±297.11)mg/L比(196.81±58.54)mg/L,(P<0.01)。BALF中白细胞、中性粒细胞和巨噬细胞增多(184.00±20.40)×10^(4)/L比(45.00±12.09)×10^(4)/L;(152.00±48.50)×10^(4)/L比(1.00±0.58)×10^(4)/L,(20.00±5.03)×10^(4)/L比(2.00±1.15)×10^(4)/L,(P值均<0.01);MSC干预后肺损伤明显减轻(4.50±1.29)分,W/D(4.53±0.15)和总蛋白水平(588.27±72.57)mg/L明显下降;BALF中的白细胞(84.00±28.53)×10^(4)/L、中性粒细胞(59.00±20.43)×10^(4)/L和巨噬细胞(8.00±3.64)×10^(4)/L明显减少。BALF和血浆中的IL-1β,IL-6和TNF-α均较健康对照组明显升高,加入MSC的干预组则可明显降低;ALI组KGF-2 mRNA和SPC mRNA明显低于对照组,干预组KGF-2 mRNA和SPC mRNA明显高于ALI组;ALI组caspase-3 mRNA和PCNA mRNA明显高于对照组,干预组caspase-3 mRNA和PCNAmRNA明显低于ALI组,差异均有统计学意义(P<0.05);病理检查结果显示ALI组肺内胶原纤维沉积�Objective To explore the protective effect of human bone marrow-derived mesenchymal stem cells(MSC)on lipopolysaccharide(LPS)induced acute lung injury(ALI).Methods This was expermental reseach.Twenty four male SPF BALF/c mice were randomly divided into three groups,control group,ALI group and ALI+MSC group.LPS(5mg/kg)was instilled into the trachea two days after the model was made.The lung tissue of the mouse was taken,and the pathological changes of the lung injury were observed by HE staining,and the lung injury score was performed.Alveolar lavage fluid(BALF)was collected,leukocytes were counted and classified by Wright-Giemsa staining.The concentration of total protein and inflammatory cytokines Interleukin 1β(IL-1β),Interleukin 6(IL-6)and tumor necrosis factor alpha(TNF-α)were determined.Lung tissue wet-to-dry ratio(W/D),RT-PCR detection of caspase-3,keratinocyte growth factor2(KGF-2),surface active protein C(SPC),proliferating cell nuclear antigen(PCNA)mRNA expression were performed.TUNNEL method was used to detect cell apoptosis in the lung.Immunohistochemistry was used to detect PCNA in the lung.Masson method was used to detect the deposition of collagen fibers in the lung;in vivo imaging of small animals traced the colonization of MSC in the lung.Results Compared with control group,lung injury score(11.75±1.71)scores vs(2.75±0.96)scores,(P<0.01),total protein concentration in BALF(1181.80±297.11)mg/L vs(196.81±58.54)mg/L(P<0.01),and W/D(5.23±0.15 vs 4.36±0.29)significantly increasedin ALI group(All P<0.01).White blood cells(184.00±20.40)×10^(4)/L vs(45.00±12.09)×10^(4)/L,neutrophils(152.00±48.50)×10^(4)/L vs(1.00±0.58)×10^(4)/L,P<0.01,macrophages(20.00±5.03 vs 2.00±1.15)×10^(4)/L,(P<0.01)in BALF were also significantly increased.After MSC intervention,lung injury significantly improved(4.50±1.29)scores,and W/D(4.53±0.15),total protein in BALF(588.27±72.57)mg/L significantly decreased.IL-1β,IL-6 and TNF-αin BALF and plasma were significantly higherin ALI groupthan in healthy contro

关 键 词：急性肺损伤 间质干细胞 模型 动物 细胞因子 

分 类 号：R563[医药卫生—呼吸系统]