BMSCs向神经元样细胞分化后对认知功能障碍大鼠学习记忆能力的影响

发布时间：2018-05-18 23:40

本文选题：骨髓间充质干细胞 + 神经元样细胞　；参考：《山西医科大学》2012年硕士论文

【摘要】：目的：观察不同传代倍数骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)向神经元样细胞分化后移植对认知功能障碍大鼠学习记忆能力的影响,为中枢神经系统退行性疾病的干细胞移植治疗提供理论和实验依据。方法：BMSCs取自健康3月龄雄性Wistar大鼠股骨,全骨髓贴壁法分离、培养、扩增、传代Wistar大鼠BMSCs。取第3代BMSCs用流式细胞仪检测CD29、CD4、CD34以鉴定纯度。分别选取第3、4、5代BMSCs用含有20%胎牛血清的lmmol/L BME预诱导24h。去掉预诱导液后,加入20ng/mL bFGF进行诱导分化48h。取诱导后的第3代BMSCs采用免疫细胞化学法鉴定神经细胞特异性抗原标记神经元特异性烯醇化酶(NSE)和神经胶质纤维酸性蛋白(GFAP)的表达。自然衰老Wistar大鼠通过Morris水迷宫筛选后随机分为4组,每组10只。对照组大鼠双侧海马注入生理盐水,3、4、5代细胞组大鼠分别注入向神经元样细胞诱导后的3、4、5代BMSCs。大鼠的学习记忆能力在移植前和实验后4周通过Morris水迷宫试验测定。采用免疫组织化学染色法观察比较移植后各组大鼠海马胆碱乙酰化酶(ChAT)阳性细胞数。结果：成功分离培养Wistar大鼠BMSCs,流式细胞仪检测表面标记为：CD29(+)、D44(+)、CD34(-),符合BMSCs的表型特征。3、4、5代BMSCs经诱导后大部分分化为神经元样细胞,诱导后的第3代BMSCs采用免疫细胞化学法鉴定神经细胞特异性抗原标记NSE表达阳性,GFAP表达阴性。Morris水迷宫试验结果：与移植前相比,对照组大鼠学习记忆成绩下降,但差异无显著性(P0.05)；3、4、5代细胞组大鼠学习记忆成绩均有提高(P0.05)。与对照组相比,3、4、5代细胞组大鼠学习记忆成绩均有提高(P0.05)。3、4、5代细胞组两两比较,差异均有显著性(P0.05)。移植后各组大鼠海马ChAT阳性结果：与对照组相比,3、4、5代细胞组大鼠海马ChAT阳性细胞数均有所增加(P0.05)。3、4、5代细胞组两两比较,差异无显著性(P0.05)。结论：3、4、5代大鼠BMSCs向神经元样细胞分化后移植均可提高认知功能障碍大鼠的学习记忆能力,且4代细胞移植效果优于3代和5代。
[Abstract]:Aim: to observe the effect of transplantation of bone marrow mesenchymal stem cell (marrow mesenchymal stem cells, BMSCs) into neuron-like cells on learning and memory ability in rats with cognitive impairment. To provide theoretical and experimental basis for stem cell transplantation treatment of central nervous system degenerative diseases. Methods BMSCs were isolated from the femur of healthy 3-month-old male Wistar rats. The whole bone marrow adherent method was used to isolate, culture, amplify and subculture BMSCs of Wistar rats. The third passage BMSCs was used to determine the purity of CD29, CD4 and CD34 by flow cytometry. The 4th and 5th generation of BMSCs were preinduced with lmmol/L BME containing 20% fetal bovine serum for 24 h. After removing the pre-induction solution, 20ng/mL bFGF was added to induce differentiation for 48 h. The expression of neuron-specific enolase (BMSCs) and glial fibrillary acidic protein (glial fibrillary acidic protein) were detected by immunocytochemistry. Natural aging Wistar rats were selected by Morris water maze and randomly divided into 4 groups with 10 rats in each group. In the control group, the rats in the control group were injected into the hippocampus of the control group and the rats in the control group were injected into the rat hippocampus of the 5th passage of normal saline. The rats in the control group were injected into the neuron-like cells induced by BMSCs for 5 passages. The learning and memory abilities of rats were measured by Morris water maze test before and 4 weeks after transplantation. The number of choline acetylase (Chat) positive cells in hippocampus of rats after transplantation was observed by immunohistochemical staining. Results: BMSCs of Wistar rats were isolated and cultured successfully. The surface markers of BMSCs were detected by flow cytometry. The results showed that the cells were differentiated into neuron-like cells after induction of BMSCs, which was in line with the phenotypic characteristics of BMSCs, and most of them were differentiated into neuron-like cells. Immunocytochemistry was used to identify the positive expression of NSE labeled with specific antigen of nerve cells. Morris water maze test showed that the learning and memory scores of the control group were lower than those before transplantation. However, there was no significant difference in the learning and memory performance of rats in the P0.05 / 4 ~ (5) passage group. Compared with the control group, the learning and memory scores of the rats in the control group were significantly higher than those in the control group. Compared with the control group, the number of ChAT positive cells in the hippocampus of the rats in the control group was significantly higher than that in the control group. There was no significant difference in the number of ChAT positive cells between the two groups. Conclusion BMSCs transplantation into neuron-like cells can improve the learning and memory ability of rats with cognitive impairment after differentiation into neuron-like cells. The effect of cell transplantation in 4th generation is better than that in 3rd and 5th generation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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