体外诱导人脐血间充质干细胞分化为类许旺细胞的进一步实验研究

发布时间：2018-04-15 08:32

本文选题：脐血 + 间充质干细胞　；参考：《蚌埠医学院》2012年硕士论文

【摘要】：背景：周围神经损伤缺损的临床治疗是外科领域尚未解决的世界难题。自体神经移植是治疗周围神经缺损的标准方法，但会造成供区神经损伤。通过组织工程化人工神经修复周围神经缺损是目前临床治疗研究的热点。人工神经的种子细胞为许旺细胞(Schwann cells,SCs)，其来源问题尚未解决。人脐血间充质干细胞(Human Umbilical Cord Blood Mesenchymal Stem Cells,HUCBMSCs)作为人骨髓间充质干细胞（Mesenchymal stem cells,MSCs）的一种替代来源，在体外诱导培养条件下可以向类SCs分化，但还需进一步进行研究。目的：本研究旨在建立HUCBMSCs的分离、培养、纯化及鉴定体系，探讨体外诱导HUCBMSCs定向分化为类SCs的方法，并优化诱导方案；进一步将诱导成功的类SCs复合去细胞神经基膜管体外共培养，示踪。方法：取人脐血标本，6%羟乙基淀粉(Hetastarch, HES)沉降红细胞，再使用人淋巴细胞分离液Ficoll（密度为1.077g/mL）分离脐血单个核细胞，在Mesencult完全培养基中培养，流式细胞仪对培养细胞进行表型测定，向成骨、成脂方向分化鉴定其多向分化的潜能；取第3代细胞进行定向诱导分化，免疫细胞化学法、RT-PCR、Western Blotting等方法对诱导后的细胞进行神经胶质细胞标志物S100b、GFAP、P75鉴定；采用反复冻融振荡洗涤法制备去细胞坐骨神经基膜管，将诱导后类SCs使用微量注射器注入去细胞基膜管中置入六孔板体外培养0、1、2W，然后取出标本行HE染色检测。结果：分离培养出的细胞低表达或不表达CD34，高表达CD44、CD73，免疫细胞化学检测发现，诱导4d后几乎所有细胞都表达神经胶质细胞标志物S100b(98±1.63%)、GFAP (95.33±2.05%)和P75(90.67±1.7%)，其中较多细胞表现出SCs经典的双极、梭形形态，同时RT-PCR、Western Blotting在基因和蛋白水平证明诱导后细胞表达神经胶质细胞标志物S100b、GFAP和P75，而未分化细胞不表达；HE染色检测结果显示去细胞基膜管中细胞可以存活并有迁移趋势。结论： 1.人脐血中可以成功分离出MSCs，HUCBMSCs在体外具有较强的增殖、自我更新能力，还具有多向分化潜能。 2. HUCBMSCs在体外可以定向分化为类许旺细胞。 3.类SCs可以在去细胞基膜管中存活、迁移，，有望成为新的种子细胞来源且为下一步人工神经移植提供依据。
[Abstract]:Background: the clinical treatment of peripheral nerve injury defect is an unsolved world problem in the field of surgery.Autologous nerve transplantation is the standard method for the treatment of peripheral nerve defect, but it can cause nerve injury in donor area.The repair of peripheral nerve defect by tissue engineering artificial nerve is the focus of clinical treatment.The seed cells of artificial nerve were Schwann cells and SCsN.Human Umbilical Cord Blood Mesenchymal Stem cells (HUCBMSCs), as an alternative source of mesenchymal stem cells (MSCs), can differentiate into SCs like stem cells in vitro, but further research is needed.Objective: to establish the system of isolation, culture, purification and identification of HUCBMSCs, to explore the method of inducing HUCBMSCs to differentiate into SCs in vitro, and to optimize the induction scheme.Furthermore, the successfully induced SCs-like neural basement tube was co-cultured in vitro.Methods: umbilical cord blood mononuclear cells were isolated from human umbilical cord blood samples from 6% Hetastarch-Hetsea (HES-) erythrocytes. The mononuclear cells from human umbilical cord blood were isolated by Ficolll (density 1.077g / mL) and cultured in Mesencult culture medium. The phenotypes of cultured cells were determined by flow cytometry (FCM).The differentiation potential of the cells in the direction of osteogenesis and adipogenesis was evaluated, and the third generation cells were selected for directional differentiation, and the glial marker S100bGFAPP75 was identified by immunocytochemistry and RT-PCRX Western Blotting.The acellular sciatic nerve basal membrane tube was prepared by repeated freeze-thaw oscillatory washing method. The induced SCs was injected into the acellular basal membrane tube with a micro syringe and cultured in vitro with a six-hole plate. The specimens were examined by HE staining.Results: after 4 days of induction, almost all of the cells expressed low or no CD34, and high expression of CD44-tir CD73. Almost all the cells expressed glial cell marker S100b(98 卤1.63 + GFAP95.33 卤2.05 and P75 + 90.67 卤1.7, among which more cells showed the classic bipolar of SCs.At the same time, the expression of glial cell markers S100bGFAP and P75 was confirmed by RT-PCR Western Blotting at the level of gene and protein. The results of HE staining showed that the cells in the acellular basement membrane tube could survive and migrate.Conclusion:1.MSCs can be successfully isolated from human umbilical cord blood and have strong proliferation, self-renewal ability and multidirectional differentiation potential in vitro.2.HUCBMSCs can differentiate into Schwan-like cells in vitro.3.SCs like can survive and migrate in acellular basement membrane tube, which may become a new seed cell source and provide the basis for the next artificial nerve transplantation.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.28

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