胚胎干细胞来源的间充质干细胞归巢及胚胎干细胞表面分子的研究

发布时间：2018-03-07 08:43

本文选题：胚胎干细胞　切入点：间充质干细胞　出处：《浙江大学》2010年博士论文　论文类型：学位论文

【摘要】：胚胎干细胞(ES)具有无限增殖能力,定向分化后可成为细胞移植的重要来源。ES定向分化后的细胞在体内尤其是病理模型体内的归巢研究对ES的医疗应用具有重要的理论和实践参考价值。在本研究中,通过ROCK抑制剂Y-27632处理并在有血清体系下单层诱导人胚胎干细胞分化为MSC(hES-MSC),在体外生物素标记hES-MSC后,将其异种移植到ICR小鼠体内。36h后,通过流式细胞术检测和组织冰冻切片的免疫组织化学(IHC)鉴定,发现了hES-MSC在ICR小鼠体内早期归巢的规律：在正常ICR小鼠体内,hES-MSC倾向于归巢到脾脏、骨髓和肺中,而几乎不在肝脏中驻留；在CCl4诱导急性肝损伤的ICR小鼠中,hES-MSC在骨髓、脾脏归巢的平均数量分别减少了64.6%和74.5%,而在损伤肝部位能发现大量归巢的hES-MSC。同时还发现在正常ICR小鼠体内随着传代次数增加,hES-MSC向脾脏、肝脏早期归巢的能力会显著性减弱,但是hES-MSC向损伤肝归巢的能力却不会受细胞代数变化的显著性影响。随后,通过RT-PCR和冰冻切片的IHC检测证明CXCR4在hES-MSC向损伤肝归巢过程中具有重要作用,而损伤肝环境会促进hES-MSC表达CXCR4和CXCR7,其中CXCR7会影响CXCR4与其配体SDF-1的结合。通过AFP和biotin的共定位,我们发现损伤肝环境还能在细胞移植早期就诱导hES-MSC表达肝向分化的指标AFP,说明hES-MSC对损伤肝的治疗作用与hES-MSC的原位肝向分化有关。在检测了TNF-α对hES-MSC归巢的影响后,发现在体外TNF-α可以促进hES-MSC表达CXCR4和CXCR7, TNF-α预处理的hES-MSC向ICR小鼠损伤肝脏早期归巢的平均数量比未处理组提高了1.4倍。综合以上的结果,可以得出这样的结论：hES-MSC不仅能定向归巢到ICR小鼠的损伤肝脏,而且还能在损伤部位进行原位肝向分化,因此hES-MSC是理想的治疗急性肝损伤的种子细胞,TNF-α预处理hES-MSC可以提高其向损伤肝早期归巢的效率从而提高治疗效果。小鼠胚胎干细胞(mES)表面分子与细胞外信号系统和胞间通讯密切相关,对mES的鉴定、自我更新及分化机理的研究具有重要意义。通过全细胞免疫获得针对mES特异性表面分子的兔单克隆抗体,利用改进的免疫共沉淀方法纯化细胞膜蛋白抗原,再结合LC-LTQ质谱鉴定和商业化抗体的western blot验证,获得了针对Glut3在mES细胞膜表面的构象表位的抗体。利用该抗体封闭Glut3可以抑制mES增殖,说明Glut3在维持mES自我更新中具有重要作用。
[Abstract]:Embryonic stem cells (es) have unlimited proliferative ability. The homing study of es cells in vivo, especially in pathological model, has important theoretical and practical reference value for the medical application of es. ROCK inhibitor Y-27632 was used to induce human embryonic stem cells to differentiate into MSCHES-MSCG in serum system. After biotin labeled hES-MSC in vitro, xenografts were transplanted into ICR mice for .36h. The early homing rule of hES-MSC in ICR mice was found by flow cytometry and immunohistochemistry identification of frozen sections. In normal ICR mice, hES-MSC tended to homing to spleen, bone marrow and lung. In ICR mice with acute liver injury induced by CCl4, HES-MSC was found in bone marrow. The average number of homing spleen decreased by 64.6% and 74.5 respectively, but a large number of homing cells were found in the injured liver. It was also found that in normal ICR mice, the ability of homing in the early stage of liver homing was significantly decreased with the increase of passage of hES-MSC to the spleen. However, the ability of hES-MSC to homing to injured liver was not affected by the significant changes of cell algebra. Subsequently, RT-PCR and frozen sections of IHC showed that CXCR4 played an important role in the homing process of hES-MSC to damaged liver. The expression of CXCR4 and CXCR7 was promoted by liver environment injury, where CXCR7 affected the binding of CXCR4 to its ligand SDF-1. We found that the injured liver environment could also induce the expression of hES-MSC in liver differentiation at the early stage of cell transplantation, indicating that the therapeutic effect of hES-MSC on the injured liver was related to the in situ liver differentiation of hES-MSC. After detecting the effect of TNF- 伪 on homing of hES-MSC, It was found that TNF- 伪 could promote the expression of CXCR4 and CXCR7 in hES-MSC in vitro. The average number of early homing of hES-MSC pretreated with TNF- 伪 to ICR mice was 1.4-fold higher than that of untreated group. It can be concluded that: HES-MSC can not only homing to the injured liver of ICR mice, but also differentiating into the liver in situ at the injured site. Therefore, hES-MSC is an ideal seed cell for the treatment of acute liver injury. TNF- 伪 pretreatment hES-MSC can improve the efficiency of homing to the early stage of liver injury and improve the therapeutic effect. The surface molecules of mouse embryonic stem cell mESs are closely related to the extracellular signaling system and intercellular communication. It is of great significance to study the mechanism of self-renewal and differentiation. Rabbit monoclonal antibodies against specific surface molecules of mES were obtained by whole-cell immunization, and cell membrane protein antigens were purified by modified co-precipitation method. Combined with LC-LTQ mass spectrometry and western blot verification of commercial antibody, the antibody against conformational epitope of Glut3 on the surface of mES cell membrane was obtained. Using this antibody to block Glut3 could inhibit mES proliferation, indicating that Glut3 plays an important role in maintaining mES self-renewal.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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