红系祖细胞

摘要： 体外生产红细胞有望缓解血源紧缺目前,红细胞和其他血液制品主要来源于志愿者外周血捐献,供者不足、感染风险、稀有血型缺乏等仍是世界性输血难题。中国科学技术大学科研人员在体外生产红细胞方向取得重要进展,他们首次建立了从人外周血单个核细胞大量扩增红系祖细胞,并高效诱导分化为成熟红细胞的实验体系,同时利用小鼠输血模型验证了该体系所产生红细胞的功能。

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.

摘要： 目的 探讨CD45阳性红系祖细胞(erythroid progenitor cell,EPC)在舌癌转移患者体内的比例及作用.方法 选取2017年1月至2018年6月就诊于河南省人民医院口腔科共40例舌癌初治患者,根据影像学和病理检查判断有无淋巴结转移,将患者分为肿瘤组(影像学和病理检查均未发现淋巴结转移者)和转移组(影像学和病理检查检测到淋巴结转移者).免疫组化检测Ki?67表达;流式细胞术检测CD45+CD71+TER119+EPC的比例,流式细胞术分选EPC;酶联免疫吸附测定(enzyme?linked immunosorbent assay,ELISA)检测上清液中白细胞介素10(interleukin?10,IL?10)和转化生长因子β(transforming growth factor?β,TGF?β)的含量;流式细胞术检测细胞内活性氧的浓度;Transwell小室进行肿瘤侵袭实验;甲基噻唑基四唑(methyl thiazolyltetrazolium,MTT)法检测人舌癌细胞CTSC?3增殖水平.结果 肿瘤组和转移组患者均为20例,对比患者年龄、性别、患病时间和肿瘤大小等一般资料,两组之间差异均无统计学意义(P>0.05);流式细胞术结果显示转移组外周血中CD45+EPC比例[(3.1±0.2)%]高于肿瘤组[(1.2±0.2)%](t=7.823,P<0.001);相关性分析的结果显示CD45+EPC比例与舌癌组织中Ki?67增殖指数呈正相关(r=0.592,P=0.006);流式细胞术结果显示转移组EPC内活性氧平均荧光强度(530.0±67.2)显著高于肿瘤组(102.1±22.9)(t=6.025,P<0.001);ELISA结果显示转移组EPC上清液中IL?10和TGF?β的质量浓度[分别为(26.9±3.7)、(6.4±0.9)μg/L]均高于肿瘤组[分别为(10.8±1.6)、(3.2±0.8)μg/L](t=3.956,P=0.003;t=2.595,P=0.027);Transwell侵袭结果显示CD45+EPC组侵袭细胞的比例[(40.3±4.4)%]显著高于对照组[(17.5±2.2)%](t=4.607,P=0.001);MTT增殖实验结果显示CD45+EPC组细胞增殖率[(52.0±3.3)%]显著高于对照组[(30.5± 1.9)%](t=5.656,P<0.001).结论 CD45+EPC在舌癌转移患者体内比例显著升高,CD45+EPC可能通过分泌免疫抑制分子和活性氧,促进肿瘤的增殖和转移.%Objective To investigate the proportion and role of CD45+erythroid progenitor cells (EPC) in patients with tongue cancer metastasis. Methods The initial treatment of tongue cancer patients (n=40) from January 2017 to June 2018 in He′nan Provincial People′s Hospital was included in this study. According to the presence or absence of lymph node metastasis, they were divided into tumor group (no lymph node metastasis was found in imaging and pathology) and metastasis group (both imaging and pathology confirmed lymph node metastasis). The expression of Ki?67 was detected by immunohistochemistry and the proportion of CD45+CD71+TER119+EPC was detected by flow cytometry. EPC was sorted by flow cytometry, interleukin?10 (IL?10) and transforming growth factor?β (TGF?β) were detected by enzyme?linked immunosorbent assay (ELISA), and reactive oxygen species (ROS) was detected by flow cytometry. Transwell was used for tumor invasion test; methyl thiazolyltetrazolium (MTT) assay was used to detect proliferation level. Results There were 20 cases in the tumor group and metastasis group. There was no significant difference between the two groups in terms of age, sex, time of onset and size of tumors. Flow cytometry showed that the ratio of CD45+EPC in peripheral blood of tumor group and metastasis group was (1.2±0.2)% and (3.1±0.2)% (t=7.823, P<0.001). Correlation analysis showed that the ratio of CD45+EPC was positively correlated with the proliferation index of Ki?67 cells (r=0.592, P=0.006). The results of flow cytometry showed that the mean fluorescence intensity (MFI) of ROS in EPC was 102.1± 22.9 in tumor group and 530.0±67.2 in metastasis group (t=6.025,P<0.001). The results of ELISA showed that the mass concentrations of IL?10 and TGF?β in EPC supernatant of tumor group were (10.8±1.6) and (3.2 ± 0.8) μg/L, respectively. The mass concentrations of IL?10 and TGF?beta in EPC supernatant of metastasis group were (26.9 ± 3.7) and (6.4 ± 0.9) μg/L, respectively (t=3.956, P=0.003; t=2.595, P=0.027). Transwell results showed that the proportion of invasive cells in the CD45+EPC group [(40.3±4.4)%] was higher than that in the control group [(17.5 ± 2.2)% ] (t=4.607, P=0.001). MTT proliferation experiment showed that the proliferation rate of the CD45+EPC group [(52.0±3.3)%] was higher than that of the control group [(30.5 ± 1.9)% ] (t=5.656, P<0.001). Conclusions The proportion of CD45+EPC in patients with tongue cancer metastasis is significantly increased. CD45+EPC can promote the proliferation and metastasis of tongue cancer by secreting immunosuppressive molecules and ROS.

摘要： Objective To discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells(EPCs)derived from umbilical cord blood(UCB)mononuclear cells(MNCs). Methods UCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing. Results With the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14-day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as(88.92±0.95)%. The percentages of cell surface markers were(86.77±9.11)%forCD71 and(64.47 ± 16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright-Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells(326.00 ± 97.96 vs 61.60 ± 20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10%DMSO were better than 5%DMSO. Additionally, 10%DMSO+2%HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective. Conclusions This non-serum culture media could effectively induced and expanded EPCs, and 10%DMSO+2%HSA+50%plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.%目的：探索含多种细胞因子无血清培养基诱导脐血单个核细胞体外分化为红系祖细胞效率及红系祖细胞的保存方法。方法利用羟乙基淀粉沉降脐血中红细胞，人淋巴细胞分离液（Ficoll）分离单个核细胞，采用含FMS样酪氨酸激酶3配体、干细胞生长因子、胰岛素样生长因子1、重组人红细胞生成素的无血清培养基进行体外诱导脐血单个核细胞向红系祖细胞分化，并将诱导的红系祖细胞用不同的冻存液进行冷冻保存，观察红系祖细胞诱导、分化能力和红系祖细胞冷冻保存效果。结果随着诱导时间延长，细胞总数明显增多，培养14 d红系祖细胞扩增约110倍，存活率为（88.92±0.95）%，红系祖细胞特异性标志CD71+细胞占（86.77±9.11）%，CD71+/CD235a+细胞占（64.47±16.67）%。诱导10 d多为中幼红细胞，可见血红蛋白表达的阳性细胞；诱导14 d开始出现晚幼红细胞，细胞沉淀呈红色。诱导7 d红系集落数为326.00±97.96，高于诱导前（61.60±20.03）。10%二甲基亚砜（DMSO）+2%人血清白蛋白保存细胞复苏后红系祖细胞存活率、回收率分别为（90.32±1.80）%、（93.66±1.87）%，将50%自体脐血浆与10%DMSO、2%人血清白蛋白联用可获得更好的保护效果。结论该无血清培养基可高效诱导、扩增红系祖细胞，10%DMSO+2%人血清白蛋白+50%自体脐血血浆可很好保存红系祖细胞。

摘要： 目的 建立和鉴定外周血中红系祖细胞来源的诱导多能干细胞(induced pluripotent stem cells,iPSCs).方法 从健康人外周血标本中分选红系祖细胞,将表达Oct4、Sox2、Lin28、L-Myc和Klf4转录因子的oriP/EBNAl附着体电转染红系祖细胞使其重编程获得iPSCs,并通过核型鉴定、碱性磷酸酶染色、RT-PCR反应、畸胎瘤形成实验及类胚体形成实验检测其干细胞多能性特性.结果 获得的iPSCs核型正常,碱性磷酸酶染色呈阳性,表达干细胞多能性基因Sox2、Oct4、Nanog和Klf4和Lin28,体内畸胎瘤形成实验可分化为内、中、外三胚层细胞,体外可形成类胚体.结论 成功建立外周血红系祖细胞来源具有多向分化潜能的iPSCs.

摘要： Objective: To investigate the dynamics changes of in vitro differentiation of erythroid progenitor cells from cord blood mononuclear cells. Methods: 0.5% methylcellulose sedimentation of cord blood erythrocytes and density gradient centrifugation in human lymphocyte separation medium were used to obtain mononuclear cells. Cells were cultured in serum-free medium containing EPO, SCF, IGF-1 and other factors to differentiate into ery⁃throid progenitor cells. The cell proliferation, survival rate and cell colony formation of cultured erythroid progeni⁃tor cells were detected. Results: The different stages erythroid progenitor cells were identified according to the ex⁃pression of the cell surface marker CD71 and CD235a. With prolonged incubation time, cell number gradually in⁃creased, cells can be amplified 140 times in 14 days. Erythroid progenitor cells can be identified by Wright-Giem⁃sa staining. Colony forming ability increased after induction, and the majority of colonies formed are erythroid colo⁃nies. Cultured erythroid progenitor cells were divided into four groups by cell surface markers' expression, which corresponding to different stages of the erythroid progenitor cells. In different time point of induction, ratio the ear⁃ly erythroid progenitor cells was decreased, while ratio of late erythroid progenitor cells was increased. Conclu⁃sion: Our current research obtained the dynamic data of in vitro differentiation of erythroid progenitor cells form cord blood mononuclear cells. Our results laid the foundation for further optimization of erythroid differentiation sys⁃ tem to inducing homogeneous erythroid progenitor cells. Our research will also be useful for the preparation of uni⁃form erythroid progenitor cells for clinical transfusion.%　　目的：探讨体外培养脐带血单个核细胞定向诱导分化为不同阶段红系祖细胞的动力学变化情况。方法：用0.5%甲基纤维素沉降脐带血红细胞及人淋巴细胞分离液密度梯度离心法得到单个核细胞，在含EPO、SCF、IGF-1等细胞因子的无血清培养体系中诱导其定向分化为红系祖细胞，观察细胞增殖、存活率、细胞集落形成情况，并检测不同阶段细胞红系特异性表面标志CD71和CD235a的表达。结果：随着培养时间的延长，细胞数逐渐增多，14 d细胞可扩增140倍左右，收集诱导后的细胞进行瑞氏吉姆萨染色，可见大量红系祖细胞，诱导后的细胞集落形成能力强，形成的克隆大部分为红系集落。诱导过程中，14 d前CD71、CD235a的表达逐渐增高。按细胞表面标志表达的不同可将诱导的细胞分为4群，分别对应红系祖细胞的不同阶段；随着诱导天数的增加，各时间点细胞对应的早期红系祖细胞群（P2、P3）比例逐渐下降，中晚期红系祖细胞群（P4、P5）的比例逐渐上升。结论：无血清培养基添加细胞因子组合的红系诱导培养体系可较好地诱导扩增红系祖细胞，流式分选可获得相对均一而处于不同分化阶段的红系祖细胞群体。获得了红系祖细胞体外分化的动力学数据，为今后进一步优化红系诱导分化体系获得均一的红系祖细胞奠定了基础，并对未来利用干细胞制备均一的红系祖细胞应用于临床治疗有一定的指导作用。

摘要： Objective To investigate the effects of erythropoietin (EPO) on the number and function of peripheral blood endothelial progenitor cells (EPCs) from rats with chronic renal failure (CRF). Methods The model of chronic renal failure was established by a two-stage 5/6nephrectomy procedure in rats. Experimental rats were randomly divided into four groups (n =7,respectively): sham operation group, CRF group, CRF rats treated with 30 U/kg EPO (low-dosage group) and with 50 U/kg EPO (high-dosage group). CRF rats were given EPO by hypodermic injection for 6 weeks, then EPCs were isolated by density gradient centrifugation from peripheral blood mononuclear cells. The ability of cell proliferation, adhesion and vasculogenesis in vitro was further observed. Results Compared to sham operation group, the ability of cell proliferation,adhesion and vasculogenesis in vitro in CRF rats was remarkably decreased (P＜0.05, respectively).Such ability was promoted significantly in dose-dependent manner by EPO treatment (P＜0.05,respectively). Conclusion EPO can improve the number and ability of endothelial progenitor cells from rats with chronic renal failure.%目的 探讨红细胞生成素(EPO)对慢性肾衰竭大鼠外周血内皮祖细胞(EPC)数量和功能的影响.方法 采用分阶段5/6肾切除制备大鼠慢性肾衰竭模型.实验动物按数字随机表法分为4组(均n=7):假手术组、慢性肾衰竭组(模型组)、30 U/kg EPO干预组(小剂量组)和50 U/kg EPO干预组(大剂量组).大鼠皮下注射EPO 6周后,取其外周血分离培养EPC,并检测EPC数量及其增殖、黏附和形成血管结构的能力.结果 与假手术组比较,慢性肾衰竭大鼠外周血EPC数量及其增殖、黏附与形成血管结构的能力均显著下降(均P＜0.05).应用EPO治疗能显著增加慢性肾衰竭大鼠外周血EPC数量(P＜0.05),改善外周血EPC增殖、黏附及形成血管结构的能力(均P＜0.05),并且呈剂量依赖性.结论 EPO可改善慢性肾衰竭大鼠外周血EPC的数量和功能.

摘要： 背景:人巨细胞病毒感染可损害造血系统,甚至造成骨髓衰竭.人巨细胞病毒感染抑制红系祖细胞增殖和分化,是否与受染红系祖细胞增殖的基因异常表达有关?目的:观察人巨细胞病毒和/或全反式维甲酸对人脐血造血干细胞向红系祖细胞定向增殖分化过程进行干预后Hoxb2、Hoxb4基因表达的变化.方法:取12例正常足月顺产新生儿断脐后的胎盘段脐血,采用造血干细胞体外培养技术及实时荧光定量聚合酶链反应方法,以人巨细胞病毒-AD169和(或)6×10-8 mol/L的全反式维甲酸持续干预人脐血造血干细胞向红系定向增殖分化过程,检测空白组、全反式维甲酸组、人巨细胞病毒组、全反式维甲酸+人巨细胞病毒组在培养第3,7,10天红系祖细胞Hoxb2、Hoxb4基因的表达情况.结果与结论:各组Hoxb2、Hoxb4基因均在增殖分化的第3天已有表达,第7天表达明显增加,第10天表达最强烈.与空白组相比,人巨细胞病毒组明显下降;与人巨细胞病毒组相比,全反式维甲酸+人巨细胞病毒组的Hoxb2、Hoxb4基因表达量明显增加(P < 0.05).提示人巨细胞病毒可能通过调控Hoxb2、Hoxb4基因异常表达而引起造血功能异常;Hoxb2、Hoxb4均与红系造血有相关性;6×10-8 mol/L全反式维甲酸能显著上调正常红系祖细胞Hoxb2、Hoxb4基因的表达,也能上调经人巨细胞病毒感染的红系祖细胞Hoxb2、Hoxb4基因的表达.

摘要： 本研究探讨全反式维甲酸(ATRA)对人脐血造血干细胞向红系定向增殖分化过程进行干预后hoxb2、hoxb4基因表达的影响.取12例正常足月顺产新生儿断脐后的胎盘段脐血,采用造血干细胞体外培养技术及FQRT-PCR方法,以6×10-8mol/L的ATRA干预人脐血造血干细胞向红系定向增殖分化的过程,进而检测空白对照组、ATRA组在培养的第3天、第7天和第10天红系祖细胞hoxb2、hoxb4基因表达情况.结果表明:本实验各组的hoxb2、hoxb4基因在第3天时开始少量表达,在第7天表达明显升高,而在第10天表达最强烈.在空白对照组中,第3天、第7天、第10天hoxb4基因相对表达量较hoxb2高.与空白对照组相比较,ATRA组Hoxb2、Hoxb4基因表达量明显增加.结论:hoxb2、hoxb4基因在脐血HSC向红系定向增殖分化过程中(体外)均有表达,说明hoxb2、hoxb4均与红系造血有相关性,且与hoxb2相比,可能hoxb4与红系造血相关性较大;6×10-8mol/L的ATRA能显著上调正常红系祖细胞hoxb2、hoxb4基因的表达.

摘要： 目的 观察复方丹参注射液(ISM)对60Coγ放射损伤小鼠骨髓红系集落形成单位(CFU-E)和红系爆式集落形成单位(BFU-E)生成的影响.方法 30只小鼠随机分为对照组(予生理盐水,不予注射)、模型组(予生理盐水,予照射)、模型+ISM给药组在60Coγ射线照射前后腹腔注射ISM,0.04ml/g,2次/d,连续注射6d,照射后第4天取股骨骨髓制备有核细胞悬液,甲基纤维素半固体培养法测定CFU-E和BFU-E集落.结果 ISM给药组CFU-E和BFU-E分别为(42.48±5.52)、(14.22±3.14)个,与模型组[分别为(16.32±2.68)、(4.62±1.88)个]相比,差异有统计学意义(P＜0.01).结论 ISM能提高放射损伤小鼠骨髓红系干细胞集落形成和保护受损小鼠骨髓造血功能的作用.

摘要： 本发明公开了泊洛沙姆在诱导造血干祖细胞增殖和/或红系分化中的用途。在造血干祖细胞增殖和/或红系分化体系中添加泊洛沙姆，能够诱导、促进造血干祖细胞增殖和/或红系分化，且增殖快，红系分化效率高。

摘要： 本发明涉及生物医学领域，具体地说是涉及一种体外诱导造血干/祖细胞向红系造血祖细胞分化的方法及其用途。本发明将基因工程与干细胞工程相结合，利用干细胞多向分化的潜能，以分泌活性WNT3a蛋白的骨髓基质细胞作为滋养层细胞，诱导造血干/祖细胞向红系造血祖细胞分化。该体外诱导体系的建立不仅为"成体干细胞可塑性"的理论提供佐证，而且可为制备临床上治疗各种原因导致的贫血、白血病等血液疾病、肿瘤放化疗的造血支持治疗、免疫系统疾病感染性疾病以及肝肾功能障碍等疾病的细胞制剂提供丰富的细胞来源。

摘要： 本发明涉及红系细胞体外诱导分化的方法，具体涉及一种促进造血干/祖细胞分化成红系细胞的诱导培养基、方法及应用。所述诱导培养基中包括天冬氨酸和/或丝氨酸。所述方法包括将造血干/祖细胞扩增培养；将扩增后的造血干/祖细胞在诱导培养基中培养，所述诱导培养基中包含天冬氨酸和/或丝氨酸。本发明通过研究发现天冬氨酸和丝氨酸对于红系细胞的诱导分化作用，从而提供了一种可以用来作为诱导分化成红系细胞的培养基以及用来诱导分化成红系细胞的方法，该培养基以及该方法可以显著促进红系细胞的诱导分化，有利于提高红系细胞的表达量以及红系细胞的数量。

摘要： 本发明公开了一种促进人造血干/祖细胞向红系分化的方法。所述方法为提供人造血干/祖细胞培养的低氧环境或者模拟低氧环境的药物。本发明发现低氧具有加速红系分化的作用，可克服现有技术分化效率低的缺点，低氧是多种干细胞的微环境，发明人首次发现其在体外促进红系分化中的作用，该作用可应用于治疗严重创伤、血液病、免疫缺陷综合征和恶性肿瘤等疾病，有助于缓解患者的贫血症状。

摘要： 本发明公开了水溶性雄黄固体分散体在制备骨髓造血干细胞/祖细胞红系分化诱导剂中的应用，所述水溶性雄黄固体分散体由包括1重量份的雄黄，1‑20重量份的高分子和0‑5重量份的表面活性剂的原料制备得到。本发明发现了水溶性雄黄固体分散体能够诱导骨髓造血干细胞/祖细胞向红细胞分化，促进骨髓细胞中红系细胞的积累，有效缓解骨髓造血干细胞/祖细胞红系分化受阻造成的红细胞数量减少；改善造血障碍导致的贫血，同时可以保护骨髓细胞免受药物的杀伤。

摘要： 本发明公开了一种缺氧条件下诱导造血干细胞定向分化为永生化红系祖细胞的方法，所述方法使用含有缺氧反应元件、CMV‑最小启动子和HPV16E6/E7基因的慢病毒表达载体转染CD34

摘要： 本发明公开了诱导造血干祖细胞增殖和红系分化的方法及其应用，其中，诱导造血干祖细胞增殖和红系分化的方法包括：利用第一培养基对造血干细胞进行第一培养，得到扩增后的造血干细胞，其中，第一培养基为添加了第一添加剂的Stemspan无血清培养基或SCGM无血清培养基，且第一添加剂为选自重组人干细胞因子、重组人促红素、白介素‑3、白介素‑6和FMS样酪氨酸激酶3的至少一种；以及利用第二培养基对上述扩增后的造血干细胞进行第二培养，得到红系祖细胞，其中，第二培养基为添加了第二添加剂的SCGM无血清培养基，且第二添加剂为选自重组人干细胞因子、胰岛素样生长因子1、重组人促红素、转鉄蛋白、地塞米松、谷胺酰胺和类脂的至少一种。

摘要： 本发明涉及一种用于长期离体维持或扩增人成红细胞、人巨核细胞‑红系祖细胞或人普通髓系祖细胞中的一种或多种的方法，所述方法包括以下步骤：在培养基中培养包含那些细胞中的一种或多种的细胞，所述培养基包含选自端锚聚合酶抑制剂、生长因子、B‑Raf激酶抑制剂和GSK‑3抑制剂中的一种或多种。

摘要： 本发明公开了一种缺氧条件下诱导造血干细胞定向分化为永生化红系祖细胞的方法，所述方法使用含有缺氧反应元件、CMV‑最小启动子和HPV16E6/E7基因的慢病毒表达载体转染CD34

摘要： 本发明涉及一种体外产生红系祖细胞的方法，该方法包括使遗传修饰或未遗传修饰的造血干细胞与包含糖皮质激素和自噬诱导剂的限定细胞培养基接触。