人胚胎干细胞来源红系细胞膜蛋白表达的研究  

A study on the expression of lineage specific membrane proteins on human embryonic stem cells-derived erythroid cells

作　　者：李晴[1] 邹庆 毛斌[1] 黄淑[1] 赖默温 潘旭[1] 孙文翠[1] 陈谊金 刘嘉馨[1] 马峰[1,2] 

机构地区：[1]中国医学科学院北京协和医学院输血研究所,四川成都610052 [2]中国医学科学院输血研究所四川新生命干细胞科技股份有限公司联合实验室

出　　处：《中国输血杂志》2017年第3期235-241,共7页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金(NSFC81356880)

摘　　要：目的探讨人胚胎干细胞(h ESCs)来源红系细胞在发育过程中其膜蛋白,包括膜表型分子、膜骨架蛋白和跨膜蛋白的表达情况。方法 h ESCs与小鼠主动脉-性腺-中肾(m AGM-S3)基质细胞共培养产生造血干祖细胞(CD34+CD45+)和GPA+细胞,挑取共培养12 d的细胞,同时磁珠分选人脐血中的CD34^+细胞,在多种特定细胞因子SCF、IL-6、IL-3、FLT3-L、TPO、EPO等诱导下,分别进行集落培养12-14 d产生红系爆发式集落形成单位(BFU-E),运用流式细胞术(FACs)、qRT-PCR和免疫荧光染色等方法检测并比较hESCs和人脐血CD34^+细胞2种来源的红系细胞膜蛋白的表达情况,并以后者作为对照组。结果 hESCs来源BFU-E的膜蛋白的表达趋势与对照组相近:其膜骨架蛋白和跨膜蛋白的基因,如SPTA、SPTB、ANK1、EPB41等基因转录水平都在逐渐增强,而一些膜蛋白如黏附分子CD49d、CD29、CD71等的表达逐渐降低,CD47基本不表达;带3蛋白(Band 3)表达量较高且与成体型血红蛋白β-globin的表达呈正相关。结论在hESCs向红系诱导生成过程中,一些膜表型分子表达在逐渐降低,而膜蛋白表达逐渐增强。Objective To investigate the changes in the expression of membrane proteins during hESC-derived erythroid differentiation,including phenotypic molecules,cytoskeletal proteins and transmembrane proteins.Methods Human embryonic stem cells and mAGM-S3 （a murine AGM-derived stromal cell line） cells were first co-cultured to produce CD34＋ CD45＋ hematopoietic stem/progenitor cells and GPA ＋ cells,which were harvested at day 12.At the same time,human umbilical cord blood CD34＋ cells （hUCB-CD34＋ cells） were purified from mononuclear cells by positive selection using the CD34 MicroBead Kit.Then these cells were cultured separately in semi-solid medium for 12-14 days with various cytokines including SCF,IL-6,IL-3,FLT3-L,TPO,EPO in order to generate burst-forming unit-erythroid for colony assay.The expression pattern of the membrane proteins of hESCs-derived erythroid cells were compared with those derived from hUCBCD34＋ cells using flow cytometry analysis and real-time polymerase chain reaction and immunochemical staining.Results The total gene expression of skeletal and membrane proteins of erythroid cells including SPTA,SPTB,EPB41 and ANK1 increased on these hESC-derived erythroblasts along with the cell maturation,while the expression of most adhesion molecules including CD49d,CD29 and CD71 gradually decreased,and CD47 showed no expression during colony culture,which was similar to those from hUCB-CD34＋ cells.The expression of band 3 is parallel to the progressive maturation labeled by the expression rate of β-globin,a marker for definitive hematopoiesis.Conclusion This study shows that while the expression of major red cell membrane proteins increases,the expression of most adhesion molecules decreases in vitro in erythroid induced production system,which helps lay a foundation for further studies on disease models associated with defect of membrane proteins during erythropoiesis.

关 键 词：人胚胎干细胞 红系细胞 膜蛋白表达 红系爆发式集落形成单位 带3蛋白 

分 类 号：R457.7[医药卫生—治疗学] R321[医药卫生—临床医学]