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背景:肺成纤维母细胞在肺组织修复和再生过程中起重要作用,但肺成纤维母细胞的分化特性尚未完全阐明。目的:观察原代培养的人肺成纤维母体细胞外分化特性。设计:以细胞为观察对象,观察性实验。单位:北京世纪坛医院呼吸科和清华大学第一附属医院中心实验室。对象:实验于2006-03/10在清华大学第一附属医院中心实验室完成。取接受一侧肺切除成人肺癌患者远离癌肿的正常组织行肺成纤维母细胞原代培养。肺组织取材经患者同意并签署知情同意书。实验经医院伦理委员会批准。DMEM(Dulbecco’s modified Eagle’smedium)培养液和碱性磷酸酶染色所需萘酚磷酸盐均购自美国Sigma公司。鼠抗人骨桥蛋白抗体购自美国R&B公司。方法:原代培养的人肺成纤维母细胞分别在成骨细胞培养基[含地塞米松1×(10-9-10-5)mol/L,维生素C50mg/L和β-磷酸甘油10mmol/L]和成脂肪细胞培养基[含15%马血清,地塞米松1×10-8mol/L和胰岛素10mg/L]作用下进行分化诱导。成骨细胞鉴定采用组织化学方法行碱性磷酸酶和钙化斑块染色同时应用Westernblotting法测定骨桥蛋白表达。油红染色鉴定脂肪细胞形成。主要观察指标:①在成骨细胞培养基诱导下,人肺成纤维母细胞向成骨细胞分化情况。②在成脂肪细胞培养基诱导下,人肺成纤维母细胞向脂肪细胞分化情况。结果:人肺成纤维母细胞在成骨细胞培养基诱导下,细胞内碱性磷酸酶表达增加,于培养第14天可见大量钙盐沉积,同时骨桥蛋白表达增加。在成脂肪细胞培养基作用第14天,部分细胞由梭形逐渐缩短变成椭圆形,油红染色显示细胞浆内脂肪小滴聚集。结论:人肺成纤维母细胞具有多分化潜能,能够在一定条件下向成骨细胞和脂肪细胞分化,此特性与骨髓间充质干细胞类似。
BACKGROUND: Lung fibroblasts play an important role in lung tissue repair and regeneration, but the differentiation characteristics of lung fibroblasts have not yet been fully elucidated. Objective: To observe the extracellular differentiation of human lung fibroblasts cultured in primary culture. Design: cell observation object, observational experiment. Unit: Department of Respiratory Medicine, Beijing Century Hospital and Tsinghua University First Affiliated Hospital Central Laboratory. Subjects: The experiment was performed at the Central Laboratory of the First Affiliated Hospital of Tsinghua University from March to March 2006. Primary lung fibroblasts were primary cultured from normal tissues of patients with lung cancer undergoing lung resection. Lung tissue samples were obtained with the patient’s consent and signed informed consent. The experiment was approved by the hospital ethics committee. DMEM (Dulbecco’s modified Eagle’s medium) medium and alkaline phosphatase required for naphthol phosphate were purchased from Sigma, USA. Mouse anti-human osteopontin antibody was purchased from American R & B company. Methods: Primary cultured human lung fibroblasts were cultured in osteoblast medium [dexamethasone 1 × (10-9-10-5) mol / L, vitamin C 50 mg / L and β-phosphoglycerate 10 mmol / L] and adipocyte medium [containing 15% horse serum, dexamethasone 1 × 10-8mol / L and insulin 10mg / L] under the action of differentiation induction. Osteoblast identification Alkaline phosphatase and calcified plaque staining were used histochemically to detect osteopontin expression by Western blotting. Oil red staining identified adipocyte formation. MAIN OUTCOME MEASURES: ① The differentiation of human lung fibroblasts into osteoblasts under the induction of osteoblast medium. ② adipogenic medium in the induction of human lung fibroblasts differentiate into fat cells. RESULTS: The expression of alkaline phosphatase increased in human lung fibroblasts induced by osteoblast medium. A large amount of calcium deposition was observed on day 14 and osteopontin expression was increased. On the 14th day of adipocyte medium function, some cells gradually shortened from spindle to oval, and oil red staining showed accumulation of fat droplets in cytoplasm. CONCLUSION: Human lung fibroblasts have pluripotency potential and can differentiate into osteoblasts and adipocytes under certain conditions. This characteristic is similar to that of bone marrow mesenchymal stem cells.