人循环纤维细胞的分离和鉴定

ISOLATION AND IDENTIFICATION OF CIRCULATING FIBROCYTES FROM HUMAN PERIPHERAL BLOOD

目的建立分离、培养人循环纤维细胞的方法,并探讨其细胞生物学特征。方法取成人外周血白细胞,经淋巴细胞分离液分离,接种于含20%胎牛血清的DMEM培养液中进行培养。对贴壁生长的成纤维样细胞进行免疫组织化学鉴定,流式细胞仪分析和电镜观察,并通过测定培养液中羟脯氨酸含量,研究其胶原合成能力。结果细胞培养9d后CD34、CD45、Ⅰ型胶原分子阳性,其中Ⅰ型胶原分子阳性细胞含量为83.5%;电镜下观察具有典型的成纤维细胞特征;培养液中羟脯氨酸含量为11.17mg/L,明显高于空白对照组8.07mg/L(P<0.01),表明培养细胞具有胶原合成能力。结论成人外周血中存在成纤维细胞的前体细胞,经体外分离、培养可分化为循环纤维细胞。

Objective To establish an effective method of isolating and culturing circulating fibrocytes from human peripheral blood and study the relationship between the expression specific molecule markers expression and the morphological characteristics. Methods Total peripheral blood leukocytes were isolated from human peripheral blood by being centrifuged over Ficoll-Paque and cultured in DMEM supplemented with 20% fetal calf serum. Adhered cells were detected with immtmocytochemistry, FCM and electron microscopy. The collagen synthesis was studied by measurement of the hydroxyproline concentration in the medium with chemical method. Results After 9 days cultue in vitro, CD34, CD45 and collagen 1 staining was positive and 83.5% of these cells secreted collagen 1 detected by FCM. Electron microscopy of circulating fibrocytes showed morphological characteristics of fibroblasts. The hydroxyprohne concentration in the medium was 11.17% mg/L, which was statistically and significantly different compared with 8.07mg/L in the control medium （ P 〈 0.01 ）. Conclusion Circulating fibrocytes could not only be isolated effectively from human peripheral blood and cultured in vitro, but also differentiate into fibroblast.