不同培养体系培养人脐血源基质细胞的探索性研究  

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

作　　者：张诚[1] 陈幸华[1] 高蕾[1] 张曦[1] 孔佩艳[1] 彭贤贵[1] 王庆余[1] 

机构地区：[1]第三军医大学附属新桥医院血液科,重庆市400037

出　　处：《实用医学杂志》2008年第2期172-175,共4页The Journal of Practical Medicine

基　　金：国家自然科学基金资助项目(编号:30070327;30670890);重庆市医学重点学科建设基金(编号:2006C028);第三军医大学新桥医院"1520人才培养工程"专项基金

摘　　要：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48h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9～14d(平均12.1d)时贴壁细胞集落开始形成,15～21d(平均19.4d)时集落数量最多,培养28d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。细胞化学染色显示为,非特异性酯酶染色法显示阳性,阳性率100%;过氧化物酶染色法显示阴性;糖原染色显示阳性,阳性率为100%;碱性磷酸酶染色显示部分阳性,阳性率26%。免疫细胞化学染色显示为,CD31显示阳性率为96%,CD68显示阳性率为95%,CD45阴性,纤维粘蛋白显示阳性率94%。结论:改良Dexter培养体系是一种理想的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。Objective To investigate the impacts of different culture systems on cultivation of human umbilical cord blood-derived stromal cells （hUCBDSCs） and observe the biological behavior of hUCBDSCs. Methods Fetal hUCBDSCs were cultured with either standard or modified Dexter-type cultures. Cell growth was observed by an inverted microscope and the morphological characteristics were detected by Wright＇s staining. The cells were then identified by cytochemistry and immunocytochemistry. Results The number of adherent cells cultured for 48 h, initial time of cellular extension, and time of primary culture in the modified cultures were superior to those in the standard cultures. The adherent cell colonies appeared by days 9 to 14 （mean 12.1 days）, reached to their maximum levels at days 15 to 21 （mean 19.4 days）, and then spread all over the bottom of the dishes by day 28. The cells were mostly differentiated into fibroblast-like cells, macrophage-like cells, and small round ceils. Cytochemistry assay revealed that the positive rate was 100% in both NSE and PAS stain, hUCBDSCs were positive in 26% of ALP stain but negative in POX. Immunocytochemistry stain revealed that the positive rate of hUCBDSCs was 96% for CD31, 95% for CD68, 0% for CD45, and 94% for Fn. Conclusions The modified Dexter-type cultures is an ideal way for cultivation of hUCBDSCs. The successful culture of hUCBDSCs in vitro provides a theoretical support for further studying their earlier clinical application.

关 键 词：脐血 人脐血源基质细胞 细胞培养技术 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]