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背景:肺脏含有多种内源性干细胞,分别为来源于内胚层或中胚层的前体细胞,阐明肺组织间充质干细胞的特性有助于了解其在肺损伤中的生物学作用。目的:观察体外分离培养的小鼠肺组织间充质干细胞生长状态、表面标志、分化功能等生物学特性,探讨其对肺损伤鼠的干预效果。设计、时间及地点:细胞学体外观察与随机对照动物体内实验,于2005-10/2007-08在解放军军事医学科学院基础医学研究所细胞生物学实验室完成。材料:清洁级三四周龄C57BL/6雄鼠,用于分离培养肺组织间充质干细胞;清洁级6~8周龄C57BL/6雌鼠20只,分为细胞移植组、模型对照组,10只/组。方法:分离雄鼠肺组织,Ⅱ型胶原酶消化后,Ficoll法离心,收集界面层细胞,贴壁法分离培养肺组织间充质干细胞,当细胞接近70%~80%汇合时消化传代。细胞移植组、模型对照组小鼠按20mg/kg腔注射马利兰抑制骨髓干细胞的迁移后,均建立博莱霉素诱导的肺纤维化模型,造模后细胞移植组尾静脉注入5×105个肺间充质干细胞,模型对照组气管内注射100μLPBS。14d后取材,制备肺组织石蜡切片。主要观察指标:体外培养的肺组织间充质干细胞的生长状态、免疫表型、基因表达、分化潜能、集落形成能力以及静脉移植减轻肺损伤效果。结果:小鼠肺组织来源的间充质干细胞为成纤维样细胞,在体外能够长期培养和快速扩增;其细胞免疫表型为Sca-1+,CD44+,CD29+,CD105+,CD54+,CD34-,CD45-,CD11b-,c-kit-,CD31-;不表达肺泡上皮细胞标志表面活性蛋白C、水通道蛋白5、clara细胞分泌蛋白,胚胎干细胞标志Oct-4和Nanog基因在培养扩增的细胞中持续表达;能够被诱导分化为脂肪细胞、成骨细胞和表达表面活性蛋白C的肺泡2型上皮样细胞;集落形成率约3%;经静脉输注后,细胞移植组肺损伤和纤维化程度较模型对照组减轻,Masson染色显示只有少量血管周围呈阳性,且经骨髓抑制处理后已接近正常肺组织。结论:体外培养的小鼠肺间充质干细胞能够快速扩增,可向脂肪细胞、成骨细胞和肺泡上皮细胞方向分化;静脉移植后有助于肺组织修复、减轻肺损伤和纤维化程度,且骨髓抑制不影响肺间充质干细胞的保护作用。
BACKGROUND: The lungs contain a variety of endogenous stem cells, which are precursors derived from endoderm or mesoderm, respectively. To clarify the characteristics of lung tissue mesenchymal stem cells may help to understand its biological role in lung injury. OBJECTIVE: To observe the biological characteristics of the growth status, surface markers and differentiation of mouse mesenchymal stem cells cultured in vitro, and to explore the intervention effect on mice with lung injury. DESIGN, TIME AND SETTING: In vitro cytology and randomized controlled animal in vivo experiments were performed at the Cell Biology Laboratory, Institute of Basic Medical Sciences, Chinese Academy of Military Medical Sciences, 2005-10 / 2007-08. MATERIALS: Twenty-four C57BL / 6 male mice of three or four weeks of age were used for isolation and culture of lung tissue mesenchymal stem cells. Twenty healthy C57BL / 6 female mice aged 6-8 weeks were divided into three groups: cell transplantation group, model control group, 10 / group. Methods: The lung tissues of male rats were isolated and digested with type Ⅱ collagenase. After centrifugation with Ficoll method, the interphase cells were harvested and the mesenchymal stem cells (MSCs) were isolated and cultured by adherent method. The cells were digested and passaged when confluent with 70% ~ 80% confluence. Cell transplantation group and model control group mice were injected with 20mg / kg marilan inhibited bone marrow stem cell migration, both bleomycin-induced pulmonary fibrosis model was established after transplantation of cell transplantation group tail vein injection of 5 × 105 lung Mesenchymal stem cells, model control group, tracheal injection of 100μL PBS. 14d after drawing, preparation of lung tissue paraffin sections. MAIN OUTCOME MEASURES: Growth status, immunophenotype, gene expression, differentiation potential, colony-forming ability of lung tissue mesenchymal stem cells cultured in vitro and the effect of vein graft to reduce lung injury. Results: The mouse lung tissue-derived mesenchymal stem cells were fibroblast-like cells that could be cultured and rapidly expanded in vitro. The immunophenotypes were Sca-1 +, CD44 +, CD29 +, CD105 +, CD54 +, CD34-, CD45-, CD11b-, c-kit-, CD31-; not expressing alveolar epithelial cell marker surfactant protein C, aquaporin 5, clara cell secretory protein, embryonic stem cell marker Oct-4 and Nanog gene in cultured expanded cells , Which can be induced to differentiate into adipocytes, osteoblasts and alveolar type 2 epithelial-like cells expressing surfactant protein C; the rate of colony formation was about 3%; after the intravenous infusion, the lung injury and fibrosis of the cell transplantation group The degree of reduction than the model control group, Masson staining showed only a small amount of blood vessels around the positive, and after treatment by bone marrow close to normal lung tissue. CONCLUSION: Mouse-derived mouse mesenchymal stem cells can rapidly proliferate in vitro and differentiate into adipocytes, osteoblasts and alveolar epithelial cells. After vein transplantation, they can help lung tissue repair, reduce lung injury and fibrosis, And bone marrow suppression does not affect the protective effect of mesenchymal stem cells.