维持体外扩增脐带血干细胞干性：下调异常升高的ROS和p38MAPKα信号

发布时间：2018-02-04 08:31

本文关键词： ROS 脐带血CD133~+细胞体外扩增 NAC p38MAPKα 脐带血CD133~+细胞体外扩增 NOD/SCID小鼠 p38MAPK ROS CD133~+细胞造血作用体外扩增　出处：《华中科技大学》2011年博士论文　论文类型：学位论文

【摘要】：第一部分细胞内ROS对脐带血CD133+细胞体外扩增特性的影响目的：使用抗氧化剂NAC调节体外扩增细胞内活性氧水平,评估ROS对脐带血CD133+细胞体外扩增特性的影响,并初步探讨其作用机制。方法：建立脐带血CD133+细胞无血清培养体系,CD133+细胞培养过程中分别采用不同浓度的抗氧化剂NAC清除细胞内ROS,检测不同时间点细胞内ROS水平,并通过CD133+细胞亚群变化、CFU及CAFC评估扩增后细胞功能。同时检测扩增后细胞增殖(CFSE)、细胞凋亡、衰老表型及相关基因p16INK4和p21WAF1的表达,探讨ROS作用机制。结果：脐带血CD133+细胞体外扩增过程中胞内ROS升高,并伴随着HSC自我更新能力损伤。抗氧化剂NAC可以有效清除胞内ROS,并且清除程度与药物剂量呈正相关。其中低剂量处理组(0.1mmol/L)的CD133+细胞扩增倍数、产生CFU细胞密度、CAFC形成能力均高于对照组(p0.05)。NAC高剂量组(1.0mmol/L)CD133+细胞扩增倍数低于对照组(p0.01),但CFU及CAFC形成能力强于对照组(p0.01)。NAC可以明显抑制凋亡率、衰老形成、及衰老相关蛋白的转录。高剂量的NAC抑制细胞增殖水平。结论：ROS参与调节CD133+细胞体外扩增,适当的降低ROS可促进扩增细胞干性的维持,过度清除ROS则抑制细胞增殖。ROS主要通过调控细胞增殖、凋亡及衰老途径发挥作用。第二部分抑制活化的p38 MAPKα促进脐带血造血干细胞体外扩增目的：应用p38MAPKα抑制剂及NOD/SCID小鼠人造血干细胞移植模型评估p38MAPKα对脐带血CD133+细胞体外扩增特性的影响,并初步探讨其作用机制。方法：建立脐带血CD133+细胞无血清无基质培养体系,CD133+细胞培养过程中采用p38MAPKα抑制剂阻断p38MAPKα活化,检测不同时间点及不同处理组细胞内p38MAPK活性,并通过CD133+细胞亚群变化、CFU、CAFC、细胞迁移率及CXCR4表达水平评估扩增后细胞特性,运用NOD/SCID小鼠人造血干细胞移植模型评估扩增后细胞长期造血重建能力。同时检测扩增后细胞CFSE增殖、细胞凋亡、衰老表型及相关基因p16INK4和p21WAF1的表达,探讨p38MAPKα作用机制。结果：细胞体外扩增过程中p38MAPKa被激活,并伴随着HSC自我更新能力损伤。p38MAPKα抑制剂抑制p38MAPKα活化后,促进了脐带血CD133+细胞体外扩增,代表早期干祖细胞的CD133+细胞亚群、CD133+CD38-细胞亚群、CD133+CXCR4+细胞亚群、CFU-GEMM及CAFC相较于对照组均明显增加(p0.01)。NOD/SCID小鼠人造血干细胞移植模型中,p38MAPKa抑制剂组的移植物植入率及移植物嵌合率均高于对照组(p0.01)。机制检测显示p38MAPKα抑制剂可以明显抑制凋亡率、衰老形成、及衰老相关蛋白的转录,促进细胞向髓系祖分化,对细胞分裂增殖无明显作用。结论：p38MAPKα参与调节CD133+细胞体外扩增,抑制p38MAPKα激活可促进HSC扩增,并维持HSC干性。主要通过抑制细胞凋亡及衰老途径发挥作用。第三部分ROS与p38MAPKα在脐带血CD133+细胞体外扩增中的相互关系的研究目的：分别单用或联合应用p38MAPKa抑制剂SB203580及抗氧化剂NAC调节体外扩增,比较与评估不同作用靶点对脐带血CD133+细胞体外扩增特性的影响,探讨ROS与p38MAPK在脐带血CD133+细胞体外扩增中的相互关系。方法：分别采用不加药物、单用p38MAPKa抑制剂SB203580、单用抗氧化剂NAC、联合应用p38MAPKα抑制剂SB203580及抗氧化剂NAC干预体外扩增,监测胞内ROS水平及p38MAPK活化状态,通过CD133+细胞亚群变化、CFU及CAFC评估并比较各处理组扩增后细胞特性差异。结果：两药联用下ROS抑制作用最强,下降到同期对照组的15.3±2.1%,SB203580处理组为52.0±7.8%,NAC组为63.4±9.0%,SB抑制效果优于NAC。p38MAPKα抑制剂及抗氧化剂NAC对p38MAPKa活化均有抑制作用。相对于初始细胞,SB组扩增效率最高,CD133+细胞扩增了21.93±1.36倍,NAC组扩增了14.50±1.19倍,对照组仅扩增了10.13±0.57倍,而药物联用组几乎没有扩增。CFU及CAFC结果均显示SB组优于NAC组。结论：p38MAPKα作为靶点调控HSC体外扩增效果优于ROS；胞内ROS调控造血生成；ROS-p38MAPK通路上并非单向传递信息,存在p38MAPK至ROS的反馈循环。
[Abstract]:The effect of ROS on the in vitro amplification of umbilical cord blood CD133+ cells in part 1
Objective: to regulate the in vitro expansion of reactive oxygen species (ROS) in vitro by using antioxidant NAC, and to evaluate the effect of ROS on the in vitro amplification characteristics of umbilical cord blood CD133+ cells, and to preliminarily explore its mechanism.
Methods: umbilical cord blood CD133+ cells in serum free medium, cultured CD133+ cells in vitro were treated by different concentration of antioxidants NAC removal of intracellular ROS, detect the level of ROS in cells at different time points, and the changes of CD133+ cell subsets, CFU cell function after amplification and CAFC amplification detection evaluation. At the same time after cell proliferation (CFSE) expression, apoptosis, senescence phenotype and related genes p16INK4 and p21WAF1, to explore the mechanism of ROS.
Results: the amplification of intracellular ROS in elevated cord blood CD133+ cells in vitro, and with HSC self-renewal ability damage. The antioxidant NAC can effectively remove intracellular ROS, and clear level is positive correlation with dosage. The low dose treatment group (0.1mmol/L) expansion of CD133+ cells, CFU cell density, CAFC formation ability were higher than the control group (P0.05.NAC) high dose group (1.0mmol/L) CD133+ cell proliferation ratio is lower than the control group (P0.01), but CFU and CAFC have strong ability to form in the control group (P0.01).NAC can inhibit the apoptosis rate of aging, transcription and senescence associated protein. High dose of NAC inhibited cell proliferation.
Conclusion: ROS is involved in the regulation of CD133+ cell expansion in vitro, decreasing ROS appropriately can promote the maintenance of the expansion cell's dry. Excessive clearance of ROS inhibits cell proliferation..ROS plays a role in regulating cell proliferation, apoptosis and senescence.
The second part inhibits activation of p38 MAPK alpha in promoting in vitro expansion of hematopoietic stem cells from umbilical cord blood
Objective: To evaluate the effect of p38MAPK alpha on the in vitro amplification characteristics of umbilical cord blood CD133+ cells by using p38MAPK alpha inhibitor and NOD/SCID mouse hematopoietic stem cell transplantation model, and preliminarily explore its mechanism.
Methods: umbilical cord blood CD133+ cells in serum free matrix culture system of cultured CD133+ cells in vitro by p38MAPK alpha inhibitor p38MAPK alpha activation, p38MAPK activity was detected at different time points and different treatment groups, and the changes of CD133+ cell subsets, CFU, CAFC, cell migration and expression of CXCR4 cells after amplification level evaluation using NOD/SCID mice artificial blood stem cell transplantation model to assess the amplified cells long-term hematopoietic reconstitution ability. At the same time detection amplified CFSE cell proliferation, apoptosis, and expression of senescence related genes p16INK4 and p21WAF1, to explore the mechanism of p38MAPK alpha.
Results: during the amplification of activated p38MAPKa cells in vitro, and with HSC self-renewal ability damage.P38MAPK alpha inhibitors inhibit p38MAPK alpha activation, promotes the umbilical cord blood CD133+ cells in vitro, representing early stem / progenitor cells of CD133+ cell subsets, CD133+CD38- cell subsets, CD133+CXCR4+ cell subsets, CFU-GEMM and CAFC compared with the control group were significantly increased (P0.01).NOD/SCID mice hematopoietic stem cells transplantation model in p38MAPKa inhibitor group graft implantation rate and graft chimerism rate were higher than the control group (P0.01). The mechanism showed that p38MAPK inhibitors can significantly inhibit the apoptosis rate of aging, transcription and senescence associated protein, promote cells to the spinal cord progenitor differentiation, had no obvious effect on cell proliferation.
Conclusion: p38MAPK alpha is involved in regulating CD133+ cell expansion in vitro, inhibiting p38MAPK alpha activation, promoting HSC amplification and maintaining HSC dry. It mainly plays a role in inhibiting cell apoptosis and aging.
The study of the relationship between the third part of ROS and p38MAPK alpha in the expansion of umbilical cord blood CD133+ cells in vitro
Purpose: used singly or combined with p38MAPKa inhibitor SB203580 and NAC antioxidant regulation in vitro, evaluate and compare the effect of different target amplification characteristics of cord blood CD133+ cells in vitro and explore the relationship between ROS and p38MAPK in umbilical cord blood CD133+ cells.
Methods: using without drugs, with p38MAPKa inhibitor SB203580, with antioxidant NAC, amplification of the combined application of p38MAPK alpha inhibitor SB203580 and antioxidant NAC intervention in vitro activation level of ROS and p38MAPK by monitoring intracellular changes of CD133+ cell subsets, CFU and CAFC to evaluate and compare the differences in cell characteristics after amplification of each treatment group.
Results: two drugs combined with ROS had the strongest inhibitory effect, decreased to the control group in the same period of 15.3 + 2.1%, 52 + 7.8% SB203580 treatment group, NAC group is 63.4 + 9%, the inhibiting effect of SB is better than NAC.p38MAPK alpha inhibitors and antioxidant NAC had inhibitory effect on activation of p38MAPKa cells in SB group. Compared with the initial, the amplification efficiency is highest, CD133+ cells was 21.93 + 1.36 times, group NAC was 14.50 + 1.19 times, the control group only was 10.13 + 0.57 times, and the drug combination group almost no amplification of.CFU and CAFC showed that the SB group than in NAC group.
Conclusion: p38MAPK alpha as a target for HSC amplification is superior to ROS in vitro, and ROS in cells regulate hematopoiesis. ROS-p38MAPK pathway is not a one-way transmission of information, and there is a feedback loop from p38MAPK to ROS.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R329

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