【作者】 邹菁；

【导师】 邹萍；

【作者基本信息】 华中科技大学 ， 内科学， 2011， 博士

【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(p<0.05)。NAC高剂量组(1.0mmol/L)CD133+细胞扩增倍数低于对照组(p<0.01),但CFU及CAFC形成能力强于对照组(p<0.01)。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(p<0.01)。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(p<0.01)。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。更多还原

【Abstract】 Object:To investigate the effect and mechanism of reactive oxygen species on the ex vivo expasion of umbilical cord blood CD133+cells.Methhod:A serum-and stroma-free culture system for umbilical cord blood CD133+cell ex vivo expansion was established. The levels of ROS, which reduced by antioxidant NAC in different concentration, were detected in several time points during the culture. HSC ability was investigated through the density of CD133+cell subgroup、CFU assay and 28day CAFC assay. The mechanism studies were performed, such as Carboxyfluorescein diacetate succinimidyl ester labeling、apoptosis assay、Senescence-associated (3 galactosidase (SA-(3-gal) staining and the expression of human p16INK4/p21WAF1Results:Accompanying with the impairment of HSC selfrenewal, the generation of intracellular ROS was increased during ex vivo expansion. The antioxidant NAC could eliminate ROS effectively according to the concentration. Otherwise, CD133+cells were increased in all groups, low-dose NAC led a higher increase than control group (p<0.05), but cell expansion was less in high-dose group (p<0.01). Furthermore, the numbers of CFU and CAFCs were hold in high levels in both of the NAC treated groups. Mechanism studies showed that NAC acted by suppressing proliferation, apoptosis and senescence.Conclusion:The ex vivo expasion of umbilical cord blood CD133+cells was regulated by the generation of intracellular ROS, which was acting through regulation of proliferation, apoptosis and senescence. HSC sternness could maintain in appropriate reduction of ROS, but over reduction could harm the proliferation of HSC. Object:To investigate the effect and mechanism of p38 MAPK on the ex vivo expasion of UCB CD 133+cells.Methhod:A serum-and stroma-free culture system for umbilical cord blood CD133+cell ex vivo expansion was established. During the culture, the activation of p38 MAPK was abrogated by SB203580, and the expression of P-P38 was detected in several time point. HSC ability was investigated through the density of CD133+cell subgroup、CFU assay、28day CAFC assay、migration rate and the expression of CXCR4. Repopulation ability was detected in NOD/SCID mice transplantation model. The mechanism studies were performed, such as Carboxyfluorescein diacetate succinimidyl ester labeling、apoptosis assay、Senescence-associated (3 galactosidase (SA-(3-gal) staining and the expression of humanp16INK4/p21WAFI.Results:Accompanying with the impairment of HSC selfrenewal, the activation of p38 MAPK was detected during the ex vivo expansion. SB203580 could abrogate the activation. Compared to control group, SB203580 enhanced expansion of early progenitors (CD133+ CD133+CD38-,CD133+CXCR4+, CFU-GEMM and CAFCs) after 7 d of culture (p<0.01). Furthermore, a robust human hematopoietic chimerism and rapid pace of engraftment were observed in non-obese diabetic severe combined immunodeficient mice, which were transplanted with SB203580-treated cells. Mechanism studies showed that SB203580 acted by suppressing apoptosis and senescence, but not by increasing proliferation and inhibiting differentiation.Conclusion:Inhibition of p38 MAPK promotes the ex vivo expansion and maintains the stemness of human HSCs during ex vivo expansion, which mainly via inhibiting of senescence and apoptosis. Object:To investigate the correlation and compare the effects of ROS and p38MAPK in the ex vivo expansion of UCB CD133+cells.Method:Besides the control group, UCB CD133+cells were cultured with NACn SB203580 or combination. The levels of ROS and expression of P-P38 were detected. The effects of expansion were evaluated by the density of CD133+cell subgroup、CFU assay and CAFC assay.Results:ROS was eliminated in all the three treated groups, a net 85%reduction of ROS was observed in combination group, while the ROS was reduced by 48%in SB203580 group and 37% in NAC group. Ferthermore, both SB203580 and NAC could abrogate the activation of p38 MAPK. Besides, the CD133+cells were expansion in SB group by 21.93±1.36 fold increase, in control group the number was 10.13±0.57; and NAC led a 14.50±1.19-fold increase. But the CD133+cells barely expansion in the combination group. In addition, the numbers of CFU and CAFCs were hold in higher levels in SB group compare to others.Conclusion:compare to elimination of ROS, Inhibition of p38MAPK producted a better effects on the ex vivo expansion of UCB CD133+cells; reactive oxygen species levels regulate hematopoiesis; ROS activate p38 MAPK, which further promotes ROS generation, forming a positive feedback loop to sustain ROS-p38 kinase signaling.更多还原