大鼠肺动脉和主动脉成纤维细胞的原代培养及生物学行为研究

目的 建立大鼠肺动脉和主动脉外膜成纤维细胞的体外分离、培养及鉴定的方法,并对其细胞形态和成纤维细胞转化成肌成纤维细胞的特征进行观察.方法 通过精分肺动脉和主动脉,剥取外膜,用10％的胎牛血清低糖DMEM培养基诱导细胞爬出生长.倒置显微镜下观察成纤维细胞形态,并对细胞进行免疫荧光鉴定.通过对α-平滑肌肌动蛋白(α-SMA)表达的观察确定传代细胞是否转化为肌成纤维细胞.结果 成纤维细胞能够迅速从剥取的外膜中爬出生长,波形蛋白细胞免疫荧光染色为阳性.主动脉成纤维细胞和肺动脉成纤维细胞形态差别大,前者为铺路石状,后者呈细长梭形.2种成纤维细胞经传代皆能转化为肌成纤维细胞,免疫荧光染色可见α-SMA大量表达,前者包围着细胞核呈条索状非定向分布,后者平行于细胞长轴呈细丝状表达.结论 该方法所获得的成纤维细胞纯度高,可在体外稳定培养.本研究首次发现了肺动脉和主动脉成纤维细胞形态学及生物行为上的差别,为细胞水平研究主动脉和肺动脉血管重塑,胶原沉积等相同或不同的病理机制提供了充足可靠的靶细胞模型.

Primary culture rat pulmonary artery and aortic fibroblasts and biological behaviour study

Objective Establishment of in vitro isolation,cultivation and identification methods of the pulmonary artery and aortic fibroblasts,and observation of their biological behaviors.Methods Finely isolated the pulmonary artery and aorta and stripped the adventitia,supplemented with 10％ fetal bovine serum (FBS),1％ streptomycin,and 1％ penicillin in low glucose DMEM for cell growth.Fibroblast morphology of the cells were observed under inverted microscope and identified by immunofluorescence.The expression of α-smooth muscle action (α-SMA) protein was observed to determine whether passage cells transform into myofibroblasts.Results Fibroblasts quickly climbed out from the adventitia and began their growth.Vimentin protein by immunofluorescence staining was positive.A great difference of morphology was exist between pulmonary artery and aortic fibroblasts:the former was cobblestone-like while the latter was thin spindle appearance.Both of their passages could transformed into myofibroblasts and had a high level of α-SMA expression under immunofluorescence.Of which former was surrounding the cell nucleus with streak-like and non-directional distribution and the latter was parallel to the long axis of the cell.Conclusions The culture method of our study can obtained fibroblasts with high purity and stabilization in vitro.We found for the first time that great differences exist between the pulmonary artery and aorta fibroblast in morphology and biological behaviors,which provide adequate and reliable model of the target cells for detecting the pathological mechanisms in pulmonary and aortic vascular remodeling or collagen deposition.