不同培养体系培养人脐血源基质细胞的探索性研究

目的 探索不同培养体系对人脐血源基质细胞原代培养的影响，并观察人脐血源基质细胞的生物学特性。方法 取产科胎儿脐带血，采用经典和改良Dexter培养体系培养人脐血源基质细胞。倒置显微镜动态观察细胞生长情况，瑞氏染色观察细胞形态特征，采用细胞化学和免疫细胞化学进行鉴定。结果 改良Dexter培养体系在48 h细胞贴壁数、细胞开始伸展时间及原代培养时间明显优于经典Dexter培养体系。原代培养9-14天(平均12.1天)时贴壁细胞集落开始形成，15-21天(平均19.4天)时集落数量最多，培养28天贴壁细胞铺满培养皿底，细胞类型以成纤维样细胞、巨噬样细胞、“小圆”类细胞为主。细胞化学染色显示：非特异性酯酶染色法(NSE)显示阳性，阳性率100％；过氧化物酶染色法(POX)显示阴性；糖原染色(PAS)显示阳性，阳性率为100％；碱性磷酸酶(ALP)染色显示部分阳性，阳性率26％。免疫细胞化学染色显示：CD31显示阳性率为96％，CD68显示阳性率为95％，CD45阴性，Fn显示阳性率94％。结论 改良Dexter培养体系是一种理想人脐血源基质细胞培养体系，人脐血源基质细胞在体外的成功培养为从一新的角度进一步研究其在临床的早日应用提供理论基础。

An exploratory study on cultivation of human umbilical cord blood-derived stromal cells with different culture systems

Objective To investigate the impact of different culture system on the culture of human umbilical cord blood-derived stromal cells(hUCBDSCs) and observe the biological behavior of hUCBDSCs. Methods The umbilical cord blood was taken from department of obstetrics. The hUCBDSCs was cultured with classical and improved Dexter-type culture. The state of cell growth was observed by inverted microscope and the morphological characters were detected by Wright's staining. The hUCBDSCs was identified by cytochemistry and immunocytochemistry. Results The number of adherent cells cultured for 48 h, the time of initial extention and the time of primary culture with improved Dexter-type culture was better than that of classical Dexter-type culture. By day 9-14 (at amedian of 12.1 days), adherent cell colonies formed and reached their maximum at 15-21 days(mean 19.4 days), by day 28, all adherent cells spreaded over the bottom of dish. By means of light microscopy, these cells were found to differentiate into three kinds of cells in culture for 28 days: fibroblast-liked cell, macrophage liked cell and small-round cells. Cytochemistry assay revealed that the positive rate reached 100 % in NSE stain and PAS stain , the hUCBDSCs by AL P stain were shown 26 % positive , but in POX stain the result was negative. Immunocytochemistry stain revealed that the positive rate of hUCBDSCs for CD31 , CD68 , CD45 , Fn , etc reached 96 % , 95 % , 0% , 94 % , respectively. Conclusion The improved Dexter-type culture was an ideal way for the culture of hUCBDSCs. The successful culture of hUCBDSCs in vitro made the foundation for further studying the application of hUCBDSCs in clinic from a new point.