摘要
背景:随着人口老龄化、医疗技术不断的更新,红细胞需求呈增加趋势,血液供应也日趋紧张。体外制备可供临床应用的血液制品成为众多专家关注的热点,而体外诱导造血干细胞向成熟红细胞分化是获得新型血源的可能途径之一。目的:探讨采用三阶段悬浮培养体系诱导外周血废弃白膜层来源造血干细胞向成熟红细胞分化及体外生产成熟红细胞的可行性。方法:经河南省红十字血液中心伦理委员会批准,采用外周血废弃白膜层为原料(献血者均知情同意),分离出CD34+细胞,以此为种子细胞,采用三阶段21 d悬浮培养体系体外诱导造血干细胞向成熟红细胞分化,分别在第4,7,9,11,13,15,17,19,21天进行细胞计数,绘制细胞生长曲线,了解细胞生长情况;在第7,11,15,19,21天进行细胞涂片和瑞氏-吉姆萨染色,显微镜下观察细胞形态学变化;在培养第7天时,采用流式细胞仪检测红系早期分化情况;在第7,11,15,17天时,采用流式细胞仪检测红系终末分化情况;在第15,17,19天时,采用流式细胞仪检测脱核率。结果与结论:①随着培养天数增加,细胞呈增加趋势,到第17天达高峰,细胞扩增约1 300倍,之后趋于平稳状态;②随着培养天数的增加,细胞从原幼红细胞向嗜碱性幼红细胞、多染幼红细胞、正染幼红细胞分化,培养至21 d时,几乎全部分化为脱核红细胞;③红系早期分化情况:IL-3R/GPA双阴性细胞占(15.8±0.21)%,其中红系爆发集落形成单位(BFU-E)占(0.98±0.21)%,红系祖细胞克隆形成单位(CFU-E)占(8.13±1.42)%;④红系终末分化情况:随着细胞培养天数的增加,GPA表达也逐渐增加,培养至第17天时,接近90%的细胞表达GPA,表明造血干细胞定向红系分化较为成功;⑤随着培养天数的增加,无核红细胞越来越多,至第19天80%以上均为无核红细胞;⑥结果表明,采用三阶段21d悬浮培养体系,体外诱导来自废弃白膜层的外周血造血干细�
BACKGROUND:With the aging of the population and the continuous updating of medical technology,the demand for red blood cells is increasing and blood supply intends to be increasingly tight.The preparation of blood products for clinical application in vitro has become a hot spot of many experts,and hematopoietic stem cells differentiating into mature red blood cells in vitro may be an approach for new blood sources.OBJECTIVE:To explore the practicability of using three-stage suspension culture system to induce the differentiation of peripheral blood hematopoietic stem cells from the abandoned buffy-coat into mature erythrocytes in vitro.METHODS:The study protocol was approved by the ethics committee of Henan Red Cross Blood Center,China.CD34+cells were isolated from peripheral blood buffy-coat,and were subsequently induced to differentiate into mature red blood cells in vitro by 21-day three-stage suspension culture system.Cell counts were performed on 4,7,9,11,13,15,17,19,and 21 days of culture,and cell growth curves were then drawn.Cell smear and May-Giemsa staining were performed on 7,11,15,19,and 21 days of culture,for cell morphology observation under a microscope.Flow cytometry was used to detect early erythroid differentiation on 7 days of culture and to detect terminal erythroid differentiation on 7,11,15,and 17 days of culture.Denucleation rate of erythroblasts was measured on 15,17,and 19 days of culture.RESULTS AND CONCLUSION:(1)With the increase of culture days,the number of cells showed an increasing trend,reaching a peak on 17 days of culture.The cells expanded to about 1 300 times,and then tended to be stable.(2)With the increase of culture days,the cells differentiated from proerythrocytes to basophilic erythrocytes,polychromatic erythrocytes and positive-stained erythrocytes,and almost all of the cells differentiated into denucleated erythrocytes on 21 days of culture.(3)The percentage of IL-3R/GPA double-negative cells was(15.8±0.21)%,of which erythroid burst-forming units accounted for(0.98±0.
作者
王姣杰
安慧娟
单泓
韩小改
别立莉
李建斌
Wang Jiaojie;An Huijuan;Shan Hong;Han Xiaogai;Bie Lili;Li Jianbin(Henan Red Cross Blood Center,Zhengzhou 450012,Henan Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2019年第29期4662-4667,共6页
Chinese Journal of Tissue Engineering Research
基金
河南省科技攻关项目(201702296)
项目负责人:王姣杰~~
关键词
造血干细胞
外周血
废弃白膜层
成熟红细胞
分化
hematopoietic stem cells
peripheral blood
abandoned buffy-coat
mature erythrocyte
differentiation