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脐带间充质干细胞临床前应用研究及经鼻粘膜细胞移植治疗大鼠脑白质损伤方法探究

发布时间:2018-11-13 13:36
【摘要】:目的培养人脐带间充质干细胞并分析细胞的生物安全性,从而为临床应用提供依据。方法剥除新鲜脐带血管,取华通式胶,采用组织贴壁法培养细胞,将不同代数的细胞(P1~P9)采用形态学、表面标志物测定、分化能力检测及增殖能力等手段进行鉴定,对P3~P6细胞进行病原学、核型、急慢性毒性、过敏及致瘤性的研究,以明确脐带间充质干细胞的安全性。结果人脐带间充质干细胞呈梭型流水状生长,并可在体外稳定扩增,细胞增殖呈‘S’型曲线;流式检测阳性标志CD90、CD44、CD73表达量均在90%以上,阴性标志表达量在P3~P6表达量较低;所培养细胞可以分化为成脂细胞、成骨细胞以及成软骨细胞。通过对细胞病原学检测,未发现细菌、真菌及其他病原微生物;采用染色体G显带法对P3~P6脐带间充质干细胞进行染色核型分析,细胞在传代过程中未发现染色体畸变;通过动物实验观察脐带间充质干细胞的安全性,注射人脐带间充质干细胞后未出现急性中毒,慢性毒性实验显示各组织器官未出现病理改变,致瘤实验在注射部位未发现肿瘤细胞,过敏实验未发现实验动物出现皮肤局部及全身的过敏反应。结论人脐带可以分离培养间充质干细胞,间充质干细胞可在体外稳定扩增培养,脐带来源的间充质干细胞具有生物安全性,可为今后临床治疗提供选择。 目的探索经鼻粘膜移植不同类型干细胞治疗大鼠脑白质损伤方法的可行性。方法培养鼠源少突胶质前体细胞、鼠源骨髓间充质干细胞、人脐带间充质干细胞。7日龄的SD大鼠制作脑白质损伤模型,共40只,随机分为四组,每组10只:(Ⅰ)鼠源少突胶质前体细胞移植组、(Ⅱ)鼠源骨髓间充质干细胞移植组、(Ⅲ)人脐带间充质干细胞移植组、(Ⅳ)对照组。10日龄模型大鼠经鼻粘膜进行细胞移植,移植前30min经鼻黏膜滴入透明质酸酶200U,细胞移植量5×105/20μl,于移植后24h、48h、72h以及7d取脑组织固定脱水,进行冰冻切片,观察细胞移植后的迁移情况。结果所培养的三种细胞生长良好,大鼠脑白质损伤模型患侧MBP表达量明显减少,,经鼻粘膜细胞移植后,模型鼠损伤部位可以发现标记的鼠源少突胶质前体细胞和鼠源骨髓间充质干细胞,并随着时间的延长迁移至损伤部位的细胞数量逐渐增多,损伤侧未发现移植的人脐带间充质干细胞。结论鼠源少突胶质前体细胞和鼠源骨髓间充质干细胞具有向损伤部位迁移的能力。经鼻粘膜细胞移植治疗脑白质损伤是一种简便、无创、可行的治疗方法。
[Abstract]:Objective to culture human umbilical cord mesenchymal stem cells and analyze their biological safety. Methods the fresh umbilical cord blood tube was stripped, the Huatong glue was taken, and the cells were cultured by tissue adherent method. The cells of different generations (P1~P9) were identified by means of morphology, surface marker measurement, differentiation ability test and proliferation ability. The etiology, karyotype, acute and chronic toxicity, allergy and tumorigenicity of P3~P6 cells were studied to determine the safety of umbilical cord mesenchymal stem cells. Results the mesenchymal stem cells of human umbilical cord were spindle-like and could be expanded stably in vitro. The proliferation of human umbilical cord mesenchymal stem cells was'S' curve. The CD90,CD44,CD73 expression of positive markers was above 90%, and the expression of negative markers was lower in P3~P6. The cultured cells could differentiate into adipoblasts, osteoblasts and chondroblasts. No bacteria, fungi and other pathogenic microorganisms were found in the cell pathogeny, and chromosome aberration was not found in the passage of P3~P6 umbilical cord mesenchymal stem cells by chromosome G banding method. The safety of umbilical cord mesenchymal stem cells was observed by animal experiments. There was no acute poisoning after injection of human umbilical cord mesenchymal stem cells. Chronic toxicity test showed no pathological changes in tissues and organs. Tumor cells were not found in the injection site in tumorigenic test, and allergic reaction in skin and whole body was not found in allergic test. Conclusion Mesenchymal stem cells can be isolated from human umbilical cord and cultured stably in vitro. Mesenchymal stem cells derived from umbilical cord have biological safety and can provide a choice for clinical treatment in the future. Objective to explore the feasibility of different types of stem cells transplanted through nasal mucosa in the treatment of brain white matter injury in rats. Methods Rat oligodendrocyte precursor cells, mouse bone marrow mesenchymal stem cells and human umbilical cord mesenchymal stem cells were cultured. The white matter injury model was made in 7-day-old SD rats. 40 rats were randomly divided into four groups. There were 10 rats in each group: (I) rodent oligodendrocyte precursor cell transplantation group, (鈪

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