摘要
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 are believed to play an important role in lung tissue repair and regenerative process. The differentiation potential of lung fibroblasts is not known very well. OBJECTIVE： To investigate the multi-differentiation capacity of human lung fibroblasts. DESIGN： An observational comparative experiment. SETTING： Department of Respiratory Medicine, Beijing Shijitan Hospital and Central Laboratory of the First Hospital of Tsinghua University. MATERIALS： This study was performed at the Central Laboratory of the First Hospital of Tsinghua University from March 2006 to October 2006. Human adult lung fibroblasts were isolated as primary cultures from resected lung of patients with lung cancer. The study was approved by hospital＇s Medical Ethics Committee, and informed consent was signed. Dulbecco＇s modified Eagle＇s medium （DMEM）, fetal calf serum （FCS）, trypsin- ethylenediamine tetraacetic acid （EDTA）, naphthol AS-MX phosphate and Fast Red TR were purchased from Sigma, USA. Mouse anti-human osteopontin antibody was from R＆D Systems China Co., Ltd. Polyvinyl difluoride （PVDF） membranes were from Bio-Rad Laboratories Inc, Hercules, CA. METHODS： Human adult lung fibroblasts were isolated as primary cultures. Human lung fibroblasts were cultured in an osteogenic medium （containing 10^-9-10^-5 mol/L dexamethasone, 50 mg/L vitamin C and 10 mmol/L β-glycerophospate） and adipogenic medium （containing 15% horse serum, 10^-8 mol/L dexamethasone and 10 mg/L insulin）, or control medium （10% FCS）. Osteoblasts were detected by alkaline phosphatese （ALP） and calcium salt staining. The expression of osteopontin was measured by Western blotting. Oil Red-O staining was used for identification of mature adipocytes. MAIN OUTCOME MEASURES： The differentiation of lung fibroblasts induced by osteogenic medium; The differentiation of lung fibroblasts induced by adipogenic medium. RESULTS： With the induction of osteogenic medium for 2 weeks, the differentiation of lung fibroblasts was induced by osteogenic and adipogenic medium, respectively. The expression of ALP and osteopontin was increased and the deposition of calcium salt was detected. After lung fibroblasts were cultured in adipogenic medium for 14 days, part of the cells gradually experienced morphological change from original spindle into oval shape. Oil Red -O staining indicated lipid drops accumulation within cytoplasma. CONCLUSION： Under certain condition, human lung fibroblasts could differentiate into osteoblasts or adipocytes that were characterized by bone marrow mesenchymal stem cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第34期6767-6770,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
the National Natural Science Foundation of China,No.30570791~~
