体外诱导分化的人脐血间充质干细胞治疗脊髓损伤的实验研究

发布时间：2018-04-22 12:13

本文选题：脐血 + 间充质干细胞　；参考：《山西医科大学》2007年硕士论文

【摘要】： 目的:探讨人脐血间充质干细胞(mesenchymal stem cells,MSCs)体外分离培养及诱导分化为神经细胞的条件;观察诱导后的MSCs源性神经细胞移植对大鼠脊髓损伤的治疗效果,为治疗中枢神经系统损伤提供实用的细胞来源。方法:1.体外分离、纯化、扩增脐血MSCs:采用Ficoll分离法分离脐血单个核细胞(mononuclear cells,MNCs),并以低糖DMEM(含10%FBS)培养基接种于6孔培养板中,胰酶消化传代。取传三代的细胞,用流式细胞仪检测细胞表面分子CD11b、CD34、CD45和CD44的表达。2.脐血MSCs的诱导分化:采用脑源性神经营养因子BDNF 10ng/ml+维甲酸RA 0.5μM+碱性成纤维生长因子bFGF 20ng/ml协同诱导培养三代的脐血MSCs定向分化。诱导7d后,用免疫荧光染色法检测星形胶质细胞特异标志GFAP及神经元特异标志MAP2的表达情况。3.体内实验:采用横切法制备大鼠脊髓横断损伤模型,实验大鼠随机分为三组:PBS组、细胞移植组、单纯手术组,每组10只。损伤术后48h,分别于损伤部位注入等量的PBS、Brdu标记的诱导后的细胞,单纯手术组损伤后不施加任何干预作为对照。采用BBB运动功能评分标准在移植术后24h及1、2、3、4、5w各时间点对大鼠进行运动功能评分。用组织学和免疫组化方法检测移植到大鼠脊髓中的Brdu阳性细胞的存活、迁移情况。结果:脐血MSCs体外培养三代后,细胞表面CD11b、CD34、CD45、CD44表达阴性。诱导分化7d后,大部分细胞的形态类似神经元,免疫荧光染色检测MAP2阳性细胞占大多数,明显多于GFAP阳性细胞。移植后2～5w,细胞移植组大鼠的后肢运动功能恢复情况均好于PBS组和单纯手术组(P＜0.05)。免疫组织化学结果显示植入的细胞可长时间在宿主脊髓中存活,并向损伤处两端迁移。结论:人脐血MSCs于体外在特定的条件下可以诱导分化为神经元样细胞为主体的神经细胞。移植脐血MSCs诱导后的神经细胞可在损伤的宿主脊髓中存活、迁移,并能促进脊髓损伤后行为和功能恢复。
[Abstract]:Objective: to investigate the conditions for the isolation, culture and differentiation of mesenchymal stem cells from human umbilical cord blood into neural cells in vitro, and to observe the therapeutic effects of induced MSCs derived neural cell transplantation on spinal cord injury in rats. To provide a useful source of cells for the treatment of central nervous system injury. Method 1: 1. In vitro isolation, purification and amplification of umbilical cord blood MSCs: mononuclear cells were isolated from cord blood mononuclear cells (MNCs) by Ficoll method, and cultured in 6-well culture plate on low glucose DMEM medium (containing 10s), and the trypsin was digested and subcultured. The expression of CD11bC34-CD45 and CD44 on the surface of the cells was detected by flow cytometry. Differentiation of cord blood MSCs: BDNF 10ng/ml retinoic acid RA 0.5 渭 M basic fibroblast growth factor bFGF 20ng/ml was used to induce MSCs directional differentiation of cord blood in three generations. After 7 days of induction, the expressions of astrocyte-specific marker GFAP and neuron-specific marker MAP2 were detected by immunofluorescence staining. In vivo experiment: the rat model of spinal cord transection was established by crosscutting method. The rats were randomly divided into three groups: PBS group, cell transplantation group, simple operation group, 10 rats in each group. 48 hours after injury, the same amount of PBS- Brdu labeled cells were injected into the injured site, and no intervention was given to the operation group after injury. The motor function score of rats was evaluated by BBB motor function score at 24 hours after transplantation and at 4 minutes and 5 weeks after transplantation. The survival and migration of Brdu positive cells transplanted into the spinal cord of rats were detected by histological and immunohistochemical methods. Results: after three passages of umbilical cord blood MSCs culture in vitro, the expression of CD11bU CD34, CD45 and CD44 was negative on the cell surface. After 7 days of differentiation, the morphology of most cells was similar to that of neurons. Immunofluorescence staining showed that the majority of MAP2 positive cells were more than those of GFAP positive cells. The recovery of hind limb motor function in the cell transplantation group was better than that in the PBS group and the simple operation group (P < 0.05). Immunohistochemical results showed that the implanted cells could survive in the host spinal cord for a long time and migrate to both ends of the injury. Conclusion: human umbilical cord blood MSCs can induce neuronal differentiation into neuron-like cells under specific conditions in vitro. Cord blood MSCs induced nerve cells could survive and migrate in the injured host spinal cord and promote the recovery of behavior and function after spinal cord injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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