脐带血造血干细胞体外生成红细胞过程中高效脱核体系的研究  

Study on an efficient denucleation system for erythropoiesis from umbilical cord blood hematopoietic stem cells in vitro

作　　者：陈晨 占启刚 盛琦 张晶晶 应燕玲[1] 章伟[1] 朱发明[1] 何吉[1] CHEN Chen;ZHAN Qigang;SHENG Qi;ZHANG Jingjing;YING Yanling;ZHANG Wei;ZHU Faming;HE Ji(Blood Center of Zhejiang Province,Hangzhou 310052,China;不详)

机构地区：[1]浙江省血液中心,杭州310052 [2]浙江省脐带血造血干细胞库

出　　处：《浙江医学》2024年第5期470-474,I0004,共6页Zhejiang Medical Journal

基　　金：浙江省基础公益研究计划项目(LTGY23H080003)。

摘　　要：目的 研究磷脂酰肌醇三羟激酶(PI3K)、组蛋白去乙酰化酶2(HDAC2)和细胞松弛素B对脐带血造血干细胞体外培养促红系脱核的作用,以期建立高效脱核体系。方法 采用磁分选富集脐带血CD34+细胞。在培养第0、4天添加干细胞因子(SCF)、IL-3、促红细胞生成素(EPO),培养第7天添加SCF、EPO,培养第11、15、18天仅添加EPO。分别于第7、11、15和18天添加PI3K(0、25、50、100 ng/mL)、HDAC2(0、60、150、300 ng/mL)、细胞松弛素B(0、25、50、75 ng/μL)3种脱核剂。采用正交设计实验分析3种脱核剂的浓度和添加时间4因素4水平对促红系脱核效果的影响,获得最佳脱核条件并作为优选组,以不加脱核剂培养作为对照组进行验证。细胞培养至第21天时,用流式细胞仪分别检测CD235+、SYTO-64-细胞,计算脱核率。使用血细胞计数仪检测RBC,ELISA法检测2,3-二磷酸甘油(2,3-DPG)、化学发光法检测ATP,观察细胞形态。结果 最佳脱核条件为培养第15天添加PI3K浓度为100 ng/mL、HDAC2浓度为150 ng/mL和松弛素B浓度为75 ng/μL。培养至第21天,优选组红细胞脱核率为(74.30±5.59)%,对照组为(28.30±14.10)%,优选组高于对照组(t=9.099,P=0.012)。培养体系获得RBC平均可达2×1010/L,红细胞ATP、2,3-DPG与正常红细胞无差异,红细胞形态与正常红细胞一致。结论 以上最佳脱核条件建立的培养体系可促进造血干细胞体外生成红细胞高效脱核。Objective To study the effects of phosphatidylinositol trihydroxykinase(PI3K),histone deacetylase 2(HDAC2) and cytochalasin B on erythroid denucleation of umbilical cord blood hematopoietic progenitor cells in vitro and establish an efficient enucleation system.Methods Cord blood CD34+cells were enriched by magnetic sorting.Cytokine stem cell factor(SCF),interleukin 3(IL-3) and erythropoietin(EPO) were added on days 0 and 4 of culture,SCF and EPO were added on day 7 of culture,and only EPO was added on days 11,15 and 18 of culture.PI3K(0 ng/mL,25 ng/mL,50 ng/mL,and 100 ng/mL),HDAC2(0 ng/mL,60 ng/mL,150 ng/mL,and 300 ng/mL) and cytorelaxin B(0 ng/μL,25 ng/μL,50 ng/μL,and 75 ng/μL) were added on the 7th,11th,15th and 18th days,respectively.Orthogonal test was used to analyze the influence of four factors and four levels of the concentration,and addition time of three kinds of nucleating agents on the effect of nucleation,to obtain the best conditions for nucleation as optimal group,while the culture without nucleating agents as the control group for verification.On the 21st day of cell culture,CD235+and SYTO-64-cells were detected by flow cytometry,and the denucleation rate was calculated.Red blood cells were counted by hemocytometer,the content of 2,3-diphosphoglycerate(2,3-DPG) was detected by ELISA,and ATP was detected by chemiluminescence assay.The cell morphology was observed.Results Statistical analysis showed that the optimal enucleation condition was as follows:the PI3K with 100 ng/mL,HDAC2 with 150 ng/mL,and Cytochalasin B with 75 ng/uL were added on the 15th day.On the 21st day of culture,the rate of erythrocyte nucleation was(74.30±5.59)% in the optimal group and(28.30±14.10)% in the control group,with the optimal group higher than the control group(t=9.099,P=0.012).There was no significant difference in the morphology and function of red blood cells compared with the control group.The number of erythrocytes reached an average of 2 × 1010/L when the cells were cultured for 21 days.Conclusion The c

关 键 词：脐带血造血干细胞 红细胞脱核 磷脂酰肌醇三羟激酶 组蛋白去乙酰化酶 细胞松弛素B 

分 类 号：R457.7[医药卫生—治疗学]