红系祖细胞在造血缺陷斑马鱼体内的移植
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摘要
目的评估红系祖细胞在先天原始造血缺陷cloche~(-/-)突变体斑马鱼体内的移植效果。方法收集绿色荧光标记红系祖细胞的转基因系zTg(gata1:EGFP)斑马鱼胚胎,制备成单细胞悬液,经流式细胞仪分选出携带绿色荧光的gata1~+细胞,利用显微注射技术将gata1~+细胞移植到42hpf的cloche~(-/-)突变体斑马鱼心脏中,采用体视荧光显微镜追踪观察移植后的红系祖细胞在cloche~(-/-)突变体斑马鱼中的表达情况。结果成功分选出携带绿色荧光的gata1~+细胞,并在移植后2 h可观察到携带绿色荧光的gata1~+细胞逐渐增殖扩散且16 h持续有绿色荧光表达。结论红系祖细胞有重建造血的潜力,为进一步研究红系祖细胞移植后的功能鉴定提供实验依据。
Objective To evaluate the transplantation effect of erythroid progenitor cells in cloche~(-/-) mutant zebrafish with congenital primary hematopoietic deficiency. Methods The embryos of zebrafish zTg(gata1:EGFP) were collected and prepared into a single cell suspension. The gata1~+ cells carrying green fluorescence were separated by flow cytometry and transplanted into the hearts of 42 hpf cloche~(-/-) mutant zebrafish by microinjection. The expression of erythroid progenitor cells in cloche~(-/-) mutant zebrafish was examined by stereo fluorescence microscopy. Results gata1~+ cells with green fluorescence were successfully selected. The proliferation of gata1~+ cells with green fluorescence was observed at 2 h after transplantation, and 16 h of continuous green fluorescence expression was observed. Conclusions Erythroid progenitor cells have a potential to reconstruct hematopoiesis and provide an experimental basis for the further study of the functional identification of erythroid progenitor cells after transplantation.
Objective To evaluate the transplantation effect of erythroid progenitor cells in cloche~(-/-) mutant zebrafish with congenital primary hematopoietic deficiency. Methods The embryos of zebrafish zTg(gata1:EGFP) were collected and prepared into a single cell suspension. The gata1~+ cells carrying green fluorescence were separated by flow cytometry and transplanted into the hearts of 42 hpf cloche~(-/-) mutant zebrafish by microinjection. The expression of erythroid progenitor cells in cloche~(-/-) mutant zebrafish was examined by stereo fluorescence microscopy. Results gata1~+ cells with green fluorescence were successfully selected. The proliferation of gata1~+ cells with green fluorescence was observed at 2 h after transplantation, and 16 h of continuous green fluorescence expression was observed. Conclusions Erythroid progenitor cells have a potential to reconstruct hematopoiesis and provide an experimental basis for the further study of the functional identification of erythroid progenitor cells after transplantation.
引文
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[3] Dalby A,Ballester-Beltran J,Lincetto C,et al.Transcription factor levels after forward programming of human pluripotent stem cells with GATA1,FLI1,and TAL1 determine megakaryocyte versus erythroid cell fate decision [J].Stem Cell Reports,2018,11(6):1462-1478.
[4] Ling T,Crispino JD,Zingariello M,et al.GATA1 insufficiencies in primary myelofibrosis and other hematopoietic disorders:consequences for therapy [J].Expert Rev Hematol,2018,11(3):169-184.
[5] Tanimura N,Miller E,Igarashi K,et al.Mechanism governing heme synthesis reveals a GATA factor/heme circuit that controls differentiation [J].EMBO Rep,2016,17(2):249-265.
[6] Kafina MD,Paw BH.Using the zebrafish as an approach to examine the mechanisms of vertebrate erythropoiesis [J].Methods Mol Biol,2018,1698:11-36.
[7] Shu LP,Zhou ZW,Zhou T,et al.Ectopic expression of Hoxb4a in hemangioblasts promotes hematopoietic development in early embryogenesis of zebrafish [J].Clin Exp Pharmacol Physiol,2015,42(12):1275-1286.
[8] 刘丰,黄慧敏,王志华,等.利用吗啉代寡核苷酸技术下调早期斑马鱼胚胎lmna基因的初步研究 [J].中国比较医学杂志,2016,26(8):85-90.
[9] Rossmann MP,Zhou Y,Zon LI.Development:For cloche the Bell Tolls [J].Curr Biol,2016,26(19):890-892.
[10] Ma N,Huang Z,Chen X,et al.Characterization of a weak allele of zebrafish cloche mutant [J].PLoS One,2011,6(11):e27540.
[11] Ulrich F,Grove C,Torres-Vazquez J,et al.Development of functional hindbrain oculomotor circuitry independent of both vascularization and neuronal activity in larval zebrafish [J].Curr Neurobiol,2016,7(2):62-73.
[12] Reischauer S,Stone OA,Villasenor A,et al.Corrigendum:Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification [J].Nature,2018,555(7697):543.
[13] Li B,Liang MY,Zhang Y,et al.Aplastic anemia associated with pregnancy:maternal and fetal complications [J].J Matern Fetal Neonatal Med,2016,29(7):1120-1124.
[14] Babushok DV,Duke JL,Xie HM,et al.Somatic HLA mutations expose the role of class I-mediated autoimmunity in aplastic anemia and its clonal complications [J].Blood Adv,2017,1(22):1900-1910.
[15] Tan WS,Lamb BW,Khetrapal P,et al.Blood transfusion requirement and not preoperative anemia are associated with perioperative complications following intracorporeal robot-assisted radical cystectomy [J].J Endourol,2017,31(2):141-148.
[16] Chennupati V,Veiga DF,Maslowski KM,et al.Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation [J].J Clin Invest,2018,128(4):1597-1614.
[17] Garcia-Carpizo V,Ruiz-Llorente S,Sarmentero J,et al.CREBBP/EP300 bromodomains are critical to sustain the GATA1/MYC regulatory axis in proliferation [J].Epigenetics Chromatin,2018,11(1):30.
[18] Liang R,Ghaffari S.Advances in understanding the mechanisms of erythropoiesis in homeostasis and disease [J].Br J Haematol,2016,174(5):661-673.
[19] Scheenstra MR,Salunkhe V,De Cuyper IM,et al.Characterization of hematopoietic GATA transcription factor expression in mouse and human dendritic cells [J].Blood Cells Mol Dis,2015,55(4):293-303.
[20] Johnson NB,Posluszny JA,He LK,et al.Perturbed MafB/GATA1 axis after burn trauma bares the potential mechanism for immune suppression and anemia of critical illness [J].J Leukoc Biol,2016,100(4):725-736.
[21] Du C,Xu Y,Yang K,et al.Estrogen promotes megakaryocyte polyploidization via estrogen receptor beta-mediated transcription of GATA1 [J].Leukemia,2017,31(4):945-956.