体外分离培养小鼠肺间充质干细胞的生物学特性及其对肺损伤干预效果(英文)

Characterization of mouse pulmonary mesenchymal stem cells isolated in vitro and the intervention effects on lung injury

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5×10^5个肺间充质干细胞，模型对照组气管内注射100μL PBS。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；能够破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： There are several types of endogenous stem/progenitor cells in lung that develop from either endoderm or mesoderm precursors. To elucidate the characteristics of the intrapulmonary stem cells will help to understand their biological behavior in lung injury. OBJECTIVE： To isolate the mesenchymal stem cells （MSCs） from mouse lung tissues, and identify their morphology and growth characteristics, cell surface antigens, differentiation potential in vitro, stem cell properties, and to investigate the protective role in bleomycin chalhmged lung. DESIGN, TIME AND SETTING： The present randomized controlled in vivo animal experiment based on in vitro observation of cytology was performed at the Laboratory of Cell Biology, Institute of Basic Medical Science, the Academy of Military Medical Science between October 2005 and August 2007. MATERIALS： Male （3-4 weeks） and female C57BL/6 （6-8 weeks） mice were used. Twenty female C57BL/6 mice were randomly and evenly divided into a pulmonary MSCs （PMSCs）-treated group and a myelosuppression group. METHODS： The lungs from male C57BL/6 mice were digested with collagenase II, followed by centrifugation over a Ficoll step. The interface fraction was collected and cultured by adherent method. When the monolayer of adherent cells reached 70%-80% confluence, the adherent cells were detected and expanded in culture medium. The mice in the PMSCs-treated and myelosuppression groups were intraperitoneally administered busulfan to inhibit the immigration of bone marrow stem cells. In addition, pulmonary fibrosis injury was induced in these mice with bleomycin. The PMSCs-treated group was intravenously administered 5×10^5 PMSCs. At the same time, the myelosuppression group received 100 μL of phosphate buffered saline. After 14 days, the lungs were taken to prepare paraffin-embedded section. MAIN OUTCOME MEASURES： Cell morphology, immunophenotype, specific markers of the induced cells, expression of gene peroxisome proliferator activated receptor γ, osteopontin, osteocalcin, prosurfactant protein C（SP-C）, Oct-4 and Nanog, histological alteration of lung tissue sections. RESULTS： MSCs were successfully isolated from mouse lung tissue. The pulmonary MSCs（PMSCs） were fibroblast-like cells, and expanded rapidly in vitro for up to 40 passages. The phenotype of the PMSCs was Sca-1^＋ ,CD44^＋ ,CD29^＋ ,CD105^＋ ,CD54^＋ ,CD34^- ,CD45^-, CD11b^-, c-kit^-, and CD31^-. They did not express alveolar epithelial cell specific markers, such as SP-C, aquaporin-5, and clara cell secretory protein. It was noticeable that stem cell markers octamer-binding transcription factor 4 （Oct-4） and Nanog were expressed continuously in culture expanded cells. They could differentiate into adipocytes, osteoblasts and alveolar epithelial cells in vitro. Colony-forming assay demonstrated that the colony forming efficiency of the PMSCs was nearly 3%. The cells from single colony were capable of differentiating to osteocytes, adipocytes and alveolar epithelial cells. Intravenous administration of PMSCs could alleviate pulmonary injury and fibrosis induced by bleomycin, despite the bone marrow was intact or not. CONCLUSION： The PMSCs are able to extensively propagate in vitro, can efficiently switch to both mesenchymal and alveolar epithelial lineages in vitro, generate adipocytes, osteocytes, and the alveolar epithelial cells, and reduce pulmonary injury and fibrosis in bleomycin challenged lung in vivo, strongly suggesting their promising applications in cellular therapy against the lung injury.