人脐血间质干细胞向神经元样细胞诱导分化和移植的实验研究

发布时间：2018-01-11 08:10

本文关键词：人脐血间质干细胞向神经元样细胞诱导分化和移植的实验研究　出处：《苏州大学》2005年硕士论文　论文类型：学位论文

【摘要】：目的1.建立体外培养、扩增人脐带血间充质干细胞(MSCs)的方法,探讨其生物学特性。2. 探讨来源于人脐血的间充质干细胞(Mesenchymal stem cells,MSCs)分化成神经元样细胞的可行性。3. 观察移植源于人脐血间质干细胞诱导分化的神经元样细胞是否可以促进大鼠脊髓损伤的行为和功能恢复。方法1.无菌条件下取正常足月剖腹产的脐带血,用淋巴细胞分离液分离脐血的单个核细胞,以偏酸性的MesencultTM作为培养基进行培养和纯化获得贴壁细胞层,测定MSCs 的生长曲线。2.培养和纯化后获得贴壁细胞,取扩增第三代后的MSCs 向神经元样细胞诱导分化。用免疫荧光标记方法检测神经元特异性标记物。3.25 只SD 大鼠随机分成移植组和对照组. 建立NYU 脊髓损伤模型移植组移植5-溴脱氧尿核苷(Brdu)标记的源于脐血的MSCs 在体外培养、扩增,并向神经元样细胞分化的细胞。用免疫荧光标记方法检测神经元特异性标记物。对照组注射PBS液.采用BBB 评分标准对移植组和照组所有大鼠术前和术后24h,1, 2, 3, 4,5 周进行运动功能评价.组织学和免疫组化分析移植细胞的定位和分化情况。结果1.来源于脐血的单个核细胞在体外用合适的培养基培养,细胞贴壁后出现破骨样及间充质样的细胞,间充质细胞为成纤维样的细胞形态,并表达MSCs相关的抗原与源于骨髓的MSCs 一致。2. 无菌条件下收集正常足月剖腹产的脐带血,经肝素抗凝,用淋巴细胞分离液分离脐血的单个核细胞,以偏酸性的MesencultTM 作为培养基进行培养和纯化,获得贴壁细胞,取扩增第三代后的MSCs向神经元样细胞诱导分化。用免疫荧光标记方法检测神经元特异性标记物。3. 免疫组化标记显示经诱导后的MSCs 表达胶质纤维酸性蛋白(GFAP),神经丝蛋白(NF)和神经元特异性烯醇化酶(NSE).移植后在脊髓病理切片中出现Brud 标记的移植细胞.神经功能检测发现一周后移植组功能恢复较对照组具有显著性差异(p0.05)。结论1.脐带血中含有的MSCs 可在体外培养、扩增,能够为实验和临床的应用提供一种新的间充质干细胞来源。2. 源于脐血的MSCs 在体外可以培养、扩增,并向神经元样细胞分化,能作为神经干细胞的来源而用于实验研究和临床应用。
[Abstract]:Objective 1. To establish a method for amplification of human umbilical cord blood mesenchymal stem cells (MSCs) in vitro. To investigate the biological characteristics of mesenchymal stem cells derived from human umbilical cord blood. The feasibility of differentiation into neuron-like cells by MSCs. 3. To observe whether the neuron-like cells derived from human umbilical cord blood mesenchymal stem cells can promote the recovery of behavior and function of spinal cord injury in rats. Methods 1. The cord blood of normal term caesarean section was collected under aseptic condition and the mononuclear cells of cord blood were separated by lymphocytes. 2. The adherent cell layer was obtained by culture and purification of partially acidic MesencultTM. The growth curve of MSCs was measured. 2. The adherent cells were obtained after culture and purification. After the third generation of MSCs was amplified to differentiate into neuron-like cells, 3.25 SD rats were randomly divided into transplantation group and control group. Establishment of NYU Spinal Cord injury Model Transplantation of 5-Bromodeoxyuridine (5-Bromodeoxyuridine). Brdu-labeled MSCs derived from umbilical cord blood was cultured in vitro. Amplification. Neurons differentiated into neuron-like cells. Neuron-specific markers were detected by immunofluorescence labeling. PBS solution was injected into the control group. BBB was used. The scoring criteria were 24 hours before and 24 hours after operation in both the transplantation group and the radiation group. The motor function was evaluated at 1, 2, 3 and 4 weeks. The localization and differentiation of transplanted cells were analyzed by histological and immunohistochemical methods. Results 1. Mononuclear cells derived from cord blood were cultured in appropriate medium in vitro. Osteoclasts and mesenchymal cells appeared after adherent, and mesenchymal cells were fibroblast-like. 2. The expression of MSCs related antigen was consistent with that of MSCs derived from bone marrow. The umbilical cord blood of normal term caesarean section was collected under aseptic condition and anticoagulant by heparin. Mononuclear cells from umbilical cord blood were isolated from lymphocytes and cultured and purified on the medium of acidic MesencultTM. The adherent cells were obtained. The third generation MSCs was induced to differentiate into neuron-like cells. The neuron-specific marker .3.The immunocytochemical staining showed that the MSCs was induced by immunocytochemistry. Expression of glial fibrillary acidic protein (GFAP). GFAP). Neurofilament protein (NFN) and neuron-specific enolase (NSE). Brud labeled transplanted cells were found in the pathological sections of spinal cord after transplantation, and the neural function test showed that the functional recovery of the transplantation group was significantly different from that of the control group one week later (p 0.05). MSCs contained in umbilical cord blood can be cultured and amplified in vitro. 2. It can provide a new mesenchymal stem cell source. 2. MSCs derived from umbilical cord blood can be cultured, expanded and differentiated into neuron-like cells in vitro. Can be used as a source of neural stem cells for experimental research and clinical applications.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R329.2

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