Mechanism by which UM171 sustains expansion of umbilical cord blood hematopoietic stem cells in vitro

doi:10.3969/j.issn.2095-4344.0898

Abstract

Abstract:

BACKGROUND: It has been reported that UM171 enables an ex vivo expansion of human hematopoietic stem cells. However, the intracellular regulatory mechanisms remain unclear.
OBJECTIVE: To investigate the compound-target interaction network of UM171 by High-Throughput Docking (HTDocking) screening technology and TargetHunter, and to outline its potential mechanisms of action.
METHODS: Using the online StemCellCKB interface, we searched the possible targets of UM171. Negative targets were excluded by validation experiments, and a surface plasmon resonance (SPR) assay was used to identify the interaction between UM171 and transforming growth factor βRI. Several important proteins which are known to regulate this pathway were investigated by western blot.
RESULTS AND CONCLUSION: StemCellCKB program automatically calculated the docking scores, which indicated the possible targets of UM171. The KD50 values were calculated as 62.5 µmol/L by SPR, indicating the high affinity between UM171 and its target protein. Western blot analysis results revealed that the expression levels of main proteins in transforming growth factor β signaling pathway, including TGF-βRI, Smad3, p-Smad3, Smad4, were increased significantly in a concentration-depend manner. Overall, UM171 can sustain the expansion of umbilical cord blood hematopoietic stem cells through the transforming growth factor β signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Fetal Blood, Hematopoietic Stem Cells, Transforming Growth Factor beta, PPAR gamma, Tissue Engineering

CLC Number:

R394.2

Xu Shi-qi, Ding Ya-hui, Zhang Yu, Ji Qing, Yang Ming, Li Ying-hui, Gao Ying-dai. Mechanism by which UM171 sustains expansion of umbilical cord blood hematopoietic stem cells in vitro[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3328-3334.

Figures/Tables 2

2.1 UM171作用于造血干细胞的流式检测结果实验中使用CD34、CD38和CD49f作为造血干细胞的富集标记来验证造血干细胞扩增效果。从人脐血中收集分离的CD34+细胞进行剂量反应分析。结果显示，UM171作用 7 d后，造血干祖细胞(CD34+CD38-)的比例和绝对数量均显著增加(图1A，B)。UM171处理后相对更原始的造血干祖细胞(CD34+CD49f+)百分比增加1倍(图1C)，同时CD34+CD49f+细胞的绝对数量也显著增加(图1D)。 2.2 StemCellCKB高通量对接(HTDocking)在线预测结果首先根据HTDocking在线预测打分结果，发现UM171的可能主要蛋白靶点为CDK2，GSK-3β，Hras，TGF-βRⅠ，erbB-2和MAPK14(图2)。 2.3 高通量对接(HTDocking)提示UM171可能抑制HDAC 在蛋白靶点预测的过程中采用与UM171具有相同生物学功能的Valproic acid(HDAC抑制剂)进行对照，根据预测发现Valproic acid(HDAC抑制剂)的主要作用靶点为CDK2，GSK-3β，Hras，erbB-2和MAPK14。通过对比分析，发现Valproic acid和UM171的预测作用靶点有90%的相似度(3A)，因此推测UM171也为一个HDAC抑制剂。然而通过酶活水平的验证结果可知，UM171对HDAC酶活的影响较小，因此UM171不是特异的HDAC抑制剂(图3B)。UM171与Valproic acid(HDAC抑制剂)预测结果中的差异蛋白质转化生长因子βRⅠ成为下一步研究的重点。 2.4 TargetHunter在线预测提示UM171可能抑制PPAR gamma 根据TargetHunter在线预测的结果可知，Chembl1801743化合物与UM171有82.98%的结构相似性(图4A，B)，因此推测Chembl1801743化合物和UM171具有相同的作用靶点。Chembl1801743是PPAR gamma的抑制剂，根据相同结构具有相似作用机制的设想，作者初步推测UM171也为PPAR gamma的抑制剂。采用PPAR gamma分析试剂盒进行验证表明，UM171对PPAR gamma并没有显著的抑制作用(图4C)。 2.5 表面等离子体共振分析根据HTDocking和TargetHunter在线预测的结果进行分析和排除后，初步选定转化生长因子βRⅠ成为下一步研究的重点。通过查阅相关文献也发现，转化生长因子βRⅠ与造血干细胞自我更新和干性维持具有较强的相关性[12，22]。而且已知55F可以与转化生长因子βRⅠ结合，通过结构模拟分析UM171与转化生长因子βRⅠ相互作用的评分与55F接近，作用相似氨基酸位点(图2，图5A)。为了确定UM171和转化生长因子βRⅠ之间的相互作用，使用了表面等离子体共振分析。重组的转化生长因子βRⅠ蛋白质通过标准氨基偶联化学过程，被偶联在CM5芯片上固定了10 min，结合在芯片上的蛋白达到6 000 RU，UM171被运行缓冲液稀释后在25 ℃以10 μL/min的流速上样。结果显示UM171与转化生长因子βRⅠ的结合具有浓度依赖性(图5B)。KD50值为62.5 µmol/L(图5C)，这意味着UM171和它的靶蛋白之间的结合能力强，不容易解离。 2. 6转化生长因子β信号通路激活为了证明UM171通过转化生长因子β信号通路扩增造血干细胞的途径，研究了几种已知的调节该途径的重要蛋白质。据报道，Smad2、Smad3和Smad4羧基端磷酸化被激活，引起细胞状态特异性调节的转录[23]。首先在细胞膜形成转化生长因子βRⅠ-转化生长因子βRⅡ复合物，然后转化生长因子βRⅡ磷酸化转化生长因子βRⅠ。激活的转化生长因子βRⅠ接下来激活Smads(p-Smad2，p-Smad3是Smad2和Smad3的激活形式)[24]。激活的Smad2和Smad3可以与Smad4形成复合物[25-26]，转移到细胞核，然后作为转录因子调节转化生长因子β通路的靶基因[20，27-28]。分析结果表明，转化生长因子βRⅠ，Smad3，p-Smad3，Smad4等主要蛋白质的表达水平显著提高，然而总的Smad2/3表达明显减少，提示UM171主要通过上调p-Smad3和Smad3/4来扩增造血干细胞。"

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