人外周血纤维细胞的分离、培养方法

目的:探索简便易行的外周血纤维细胞体外分离、培养方法极其生物学特征与功能.方法:采用Ficoll密度梯度离心分离法分离成人外周血,所获得的白细胞在一定条件要求下体外培养,采用流式细胞技术、细胞免疫荧光染色等对贴壁生长的成纤维样细胞进行鉴定,在扫描电镜下进一步观察其形态结构.结果:培养至第14天时,外周血纤维细胞开始分化成熟.血液来源的取材、首次换液的时间、细胞的接种密度、血清浓度等多种因素均会对外周血纤维细胞的培养造成影响.免疫荧光染色结果显示培养至12天时CD34和COL Ⅰ均为强阳性表达,继续培养至28天时,血液来源的细胞表面抗原CD34发生明显丢失,免疫荧光染色几乎不能显色；相反COL Ⅰ持续表达阳性,取培养14天的贴壁细胞,经流式细胞仪分析,Col Ⅰ+细胞为81.6％,显示PBFCs不断向成纤维细胞分化的特性.结论:采用密度梯度离心法配合适当的培养条件,成人外周血中存在的前体细胞经体外分离、培养可分化为外用血纤维细胞,并保持其生物学特性.

Isolation And Cell Culture of Human Peripheral Blood Fibrocytes

Objectives:To establish a simple and efficient method for isolating and culturing peripheral blood fibrocytes in vitro,study the functions and relationship between the expression specific molecule makers expression and the morphological characteristics.Methods:Total peripheral blood leukocytes were isolated from human peripheral blood by centrifugation over Ficoll-Paque.Adhered fibroblast-like cells were detected by immunocytochemis-try,flow cytometry and electron microscopy.Results:Cultured to day 14,pe-ripheral blood mature fiber cells begin to differentiate.The source of blood draw,the first exchange of fluid impact on the culture of the cell inoculation density,serum concentration,and other factors will peripheral blood fibrocytes.Immunocytochemical staining of PBFCs showed that after 14 days,CD34+ and COLⅠ+were strongly expressed,continuously cultured to 28 days,most CD34+ lost,oppositely,COLⅠ+ had strongly expressed.The result showed that PBFCs differentiated into fibroblasts sustainably.Conclusions:The density gra-dient centrifugation with the appropriate culture conditions,the existence of precursor cells in adult peripheral blood were isolated,cul-tured in vitro to differentiate into the peripheral blood fibroblast,and maintain their biological properties.