人胚胎干细胞来源红系细胞膜蛋白表达的研究
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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 h ESC-derived erythroid differentiation,including phenotypic molecules,cytoskeletal proteins and transmembrane proteins.Methods Human embryonic stem cells and m AGM-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(h UCB-CD34+cells) were purified from mononuclear cells by positive selection using the CD34 Micro Bead 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 h ESCs-derived erythroid cells were compared with those derived from h UCBCD34+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 h ESC-derived erythroblasts along with the cell maturation,while the expression of most adhesion molecules including CD49 d,CD29 and CD71 gradually decreased,and CD47 showed no expression during colony culture,which was similar to those from h UCB-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.
Objective To investigate the changes in the expression of membrane proteins during h ESC-derived erythroid differentiation,including phenotypic molecules,cytoskeletal proteins and transmembrane proteins.Methods Human embryonic stem cells and m AGM-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(h UCB-CD34+cells) were purified from mononuclear cells by positive selection using the CD34 Micro Bead 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 h ESCs-derived erythroid cells were compared with those derived from h UCBCD34+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 h ESC-derived erythroblasts along with the cell maturation,while the expression of most adhesion molecules including CD49 d,CD29 and CD71 gradually decreased,and CD47 showed no expression during colony culture,which was similar to those from h UCB-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.
引文
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[20]郁金凤,孙文翠,路旭琳,等.人胚胎干细胞高效诱导产生巨噬细胞方法的建立.中国输血杂志,2015,28(5):482-487.
[21]Zhang J,Randall MS,Loyd MR,et al.Mitochondrial clearance is regulated by Atg7-dependent and-independent Mechaisms during reticulocyte maturation.Blood,2009,114(1):157-164.
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[23]Chen K,Liu J,Heck S,et al.Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression.Proc Natl Acad Sci USA,2009,106(41):17413-17418.
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[25]Mao B,Shu H,Lu XL,et al.Early development of definitive erythroblasts from human pluripotent stem cells defined by expression of glycophorin A(CD235a),CD34 and CD36.Stem Cell Reports,2016,7(5):869-883.
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[27]Eber S,Lux SE.Hereditary spherocytosis--defects in proteins that connect the membrane skeleton to the lipid bilayer.Semin Hematol,2004,41(2):118-141.
[2]von Lindern M,Zauner W,Mellitzer G,et al.The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro.Blood,1999,94(2):550-559.
[3]Wessely O,Bauer A,Quang CT,et al.A novel way to induce erythroid progenitor self renewal:Cooperation of c-Kit with the erythropoietin receptor.Biological Chemistry,1999,380(2):187-202.
[4]Carotta S,Pilat S,Mairhofer A,et al.Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells.Blood,2004,104(6):1873-1880.
[5]Chang KH,Nelson AM,Cao H,et al.Definitive-like erythroid cells derived from human embryonic stem cells coexpress high levels of embryonic and fetal globins with little or no adult globin.Blood,2006,108(5):1515-1523.
[6]Lu SJ,Feng Q,Park JS,et al.Biologic properties and enucleation of red blood cells from human embryonic stem cells.Blood,2008,112(12):4475-4484.
[7]Ma F,Ebihara Y,Umeda K,et al.Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis.Proc Natl Acad Sci USA,2008,105(35):13087-13092.
[8]Stephenson JR,Axelrad AA,Mc Leod DL,et al.Induction of colonies of hemoglobin-synthesizing cells by erythropoietin in vitro.Proc Natl Acad Sci USA,1971,68(7):1542-1546.
[9]Dover GJ,Chan T,Sieber F.Fetal hemoglobin production in cultures of primitive and mature human erythroid progenitors:differentiation affects the quantity of fetal hemoglobin produced per fetalhemoglobin-containing cell.Blood,1983,61(6):1242-1246.
[10]Blikstad I,Nelson WJ,Moon RT,et al.Synthesis and assembly of spectrin during avian erythropoiesis:stoichiometric assembly but unequal synthesis of alpha and beta spectrin.Cell,1983,32(4):1081-1091.
[11]Chen KL,Liu J,Heck S,et al.Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.Proc Natl Acad Sci USA,2009,106(41):17413-17418.
[12]Liu J,Mohandas N,An X.Membrane assembly during erythropoiesis.Curr Opin Hematol,2011,18(3):133-138.
[13]Waugh RE,Mc Kenney JB,Bauserman RG,et al.Surface area and volume changes during maturation of reticulocytes in the circulation of the baboon.J Lab Clin Med,1997,129(5):527-535.
[14]Zhang J,Randall MS,Loyd MR,et al.Mitochondrial clearance is regulated by Atg7-dependent and independent mechanisms during reticulocyte maturation.Blood,2009,114(1):157-164.
[15]Kundu MI,Lindsten T,Yang CY,et al.Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation.Blood,2008,112(4):1493-1502.
[16]Liu J,Guo X,Mohandas N,et al.Membrane remodeling during reticulocyte maturation.Blood,2010,115(10):2021-2027.
[17]Keerthivasan G,Small S,Liu H,et al.Vesicle trafficking plays a novel role in erythroblast enucleation.Blood,2010,116(17):3331-3340.
[18]Sandoval H,Thiagarajan P,Dasgupta SK,et al.Essential role for Nix in autophagic maturation of erythroid cells.Nature,2008,454(7201):232-235.
[19]Liu J,Zhang J,Ginzburg Y,et al.Quantitative analysis of murine terminal erythroid differentiation in vivo:novel method to study normal and disordered erythropoiesis.Blood,2013,121(8):e43-e49.
[20]郁金凤,孙文翠,路旭琳,等.人胚胎干细胞高效诱导产生巨噬细胞方法的建立.中国输血杂志,2015,28(5):482-487.
[21]Zhang J,Randall MS,Loyd MR,et al.Mitochondrial clearance is regulated by Atg7-dependent and-independent Mechaisms during reticulocyte maturation.Blood,2009,114(1):157-164.
[22]Liu J,Mohandas N,An X Membrane assembly during erythropoiesis.Cur Opin Hematol,2011,18(3):133-138.
[23]Chen K,Liu J,Heck S,et al.Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression.Proc Natl Acad Sci USA,2009,106(41):17413-17418.
[24]Southcott MJ,Tanner MJ,Anstee DJ.The expression of human blood group antigens during erythropoiesis in a cell culture system.Blood,1999,93(12):4425-4435.
[25]Mao B,Shu H,Lu XL,et al.Early development of definitive erythroblasts from human pluripotent stem cells defined by expression of glycophorin A(CD235a),CD34 and CD36.Stem Cell Reports,2016,7(5):869-883.
[26]Low PS,Allen DP,Zioncheck TF,et al.Tyrosin phosphorylation of Band 3 inhibits peripheral protein binding.J.Biol.Chem,1987,262(10):4592-4596.
[27]Eber S,Lux SE.Hereditary spherocytosis--defects in proteins that connect the membrane skeleton to the lipid bilayer.Semin Hematol,2004,41(2):118-141.