骨髓间充质干细胞体外定向分化为神经样细胞的研究

发布时间：2018-01-16 04:17

本文关键词：骨髓间充质干细胞体外定向分化为神经样细胞的研究　出处：《广州医学院》2010年硕士论文　论文类型：学位论文

【摘要】：脊髓损伤(spinal cord injury, SCI)后所引起的神经源性膀胱症状严重影响患者的生活质量,但目前尚无有效的治疗手段,并且多数研究集中在尿流动力学及神经功能损伤等方面。随着近年来干细胞研究的突飞猛进,干细胞移植用于治疗血液系统恶性肿瘤、神经系统自身免疫疾病并取得有效成果,甚至已经应用于临床,这使通过干细胞联合组织工程移植增加脊髓神经数量、减少胶质瘢痕和空洞的形成成为可能,采用干细胞接种至组织工程进行移植可望成为一种有效治疗SCI后神经源性膀胱的新方法。然而,在组织工程构建中选择合适的种子细胞成为治疗的重要环节。全能干细胞和多能干细胞具有高度自我更新能力和多向分化的潜能,是组织工程中重要的靶细胞。胚胎干细胞移植存在伦理道德和免疫排斥等问题,神经干细胞、嗅鞘细胞及雪旺细胞的来源均有限,且取材不便,很难应用于临床。BMSCs在体外具有强大的增殖能力,而且可以在不同的理化环境和细胞因子的诱导下向骨、脂肪、腱和肌肉等组织分化,还可以跨胚胎分化为神经组织,展示出良好的研究和应前用景。实验目的: 通过对SD大鼠BMSCs的体外分离纯化和培养扩增,采用免疫荧光染色法对BMSCs进行形态学观察及表型鉴定,并利用其生物学特性进行Nestin免疫细胞化学分析、诱导分化及分化鉴定,探讨BMSCs定向分化为神经样细胞的潜能,为利用BMSCs治疗SCI所引起的神经源性膀胱提供一种理想的种子细胞和一定的理论依据。实验方法: 1.采用全骨髓贴壁分离法体外提取、分离及纯化大鼠BMSCs,并进行传代扩增; 2.绘制BMSCs的生长曲线,并进行免疫荧光染色观察其形态结构; 3.流式细胞分析仪检测细胞表面抗原CD29、CD34、CD105的阳性表达率; 4.在成骨细胞诱导剂和神经细胞诱导剂条件下定向诱导BMSCs向成骨细胞和神经样细胞分化,并分别行茜素红、Nestin免疫细胞化学染色鉴定; 5.将诱导形成的神经球样细胞团进一步培养分化,并进行MAP-2、GFAP免疫细胞化学染色鉴定分化形成的神经元、星形胶质细胞结构。实验结果: 1.全骨髓贴壁分离法获得的BMSCs在接种后24～36小时贴壁,梭形,核质比大,呈漩涡状生长,增殖能力强; 2.细胞生长经过潜伏适应期、对数增长期、平台期阶段;免疫荧光染色后在荧光显微镜下可见全部细胞均呈绿色荧光,细胞为梭形或多边形,细胞核未染荧光,保持了良好的形态; 3.流式细胞分析仪显示P3～P5代细胞表面抗原CD29、CD105表达阳性,而CD34表达阴性; 4.在两种不同诱导剂诱导下BMSCs能分化形成成骨细胞和神经样细胞并表达相应的特异性抗原; 5.进一步培养分化所形成的细胞能表达神经元标志物MAP-2或神经胶质细胞标志物GFAP。结论: 1.全骨髓贴壁分离法可分离出较纯的BMSCs,经培养扩增后能获取大量的BMSCs,细胞呈贴壁生长,增殖能力强; 2.BMSCs具有多向分化的潜能,在体外可诱导分化为成骨细胞及神经样细胞; 3.分化形成的神经球样细胞能进一步分化为神经元和神经胶质细胞,是干细胞移植治疗的理想种子细胞,具有极大的应用前景。
[Abstract]:Spinal cord injury (spinal cord, injury, SCI) of neurogenic bladder symptoms caused by the serious influence the quality of life of patients, but there is no effective treatment, and most studies concentrated flow dynamics and nerve function injury in urine. Along with the rapid development of stem cell research, stem cell transplantation for the treatment of blood system malignant tumor, autoimmune diseases of the nervous system and achieved effective results, even has been used in clinical, which made by stem cells and tissue engineering transplantation increases the number of spinal nerves, the reduction of glial scar formation is possible, the stem cells were inoculated to tissue engineering transplant may be a new method for neurogenic bladder effectively after the treatment of SCI. However, in the construction of tissue engineering in the selection of suitable seed cells has become an important part of treatment. All stem cells and pluripotent stem Cells have self-renewal and multilineage differentiation potential, is an important target cells in tissue engineering. Embryonic stem cells exist ethical issues and immune rejection, neural stem cells, with limited sources of olfactory ensheathing cells and Schwann cells, and the sampling is inconvenient, it is difficult to apply to clinical.BMSCs has strong proliferation ability in vitro, but also fat to the bone, induced by different physical and chemical environment of cytokines, tendon and muscle tissue differentiation, can also cross the embryonic differentiation into nerve tissue, show good prospect of research and application.
Objective:
Purified and cultured by SD amplification of rat BMSCs in vitro, morphology and phenotype of BMSCs by immunofluorescence staining, and analyzed by Nestin immunocytochemistry using its biological characteristics, differentiation and differentiation, to explore the differentiation of BMSCs into neuron like cell potential, provides an ideal seed cell and a theoretical basis of neurogenic bladder caused by the use of BMSCs for the treatment of SCI.
Experimental methods:
1. the rat BMSCs was isolated and purified by the method of full bone marrow adherence separation in vitro.
2. the growth curve of BMSCs was plotted and the morphological structure was observed by immunofluorescence staining.
3. flow cytometry was used to detect the positive expression of cell surface antigen CD29, CD34 and CD105.
4., BMSCs was induced to differentiate into osteoblasts and neuron like cells under the condition of osteoblast inducers and nerve cell inducers. They were identified by alizarin red and Nestin immunocytochemical staining.
5., the induced neuron like cell clusters were further cultured and differentiated, and MAP-2 and GFAP immunocytochemical staining were used to identify the differentiated neurons and astrocytes.
Experimental results:
1. the BMSCs obtained by the full bone marrow adherence separation method was adhered to the wall 24~36 hours after inoculation, spindle shape, large nuclear substance ratio, whirlpool like growth and strong proliferation ability.
2., cell growth is in latent adaptation period, logarithmic growth stage and plateau stage. After immunofluorescence staining, all cells in the fluorescence microscope showed green fluorescence. The cells were spindle shaped or polygonal, and the nuclei were not fluorescent, and maintained a good morphology.
The 3. flow cytometry showed that the cell surface antigen of P3 to P5 was CD29, and the expression of CD105 was positive, but the expression of CD34 was negative.
4. under the induction of two different inducers, BMSCs can differentiate into osteoblasts and nerve like cells and express corresponding specific antigens.
5. the cells formed by further differentiation can express the neuron marker MAP-2 or the glial cell marker GFAP.
Conclusion:
1. the total bone marrow adherent separation method can separate the pure BMSCs. After culture amplification, a large number of BMSCs can be obtained. The cells are adhered to the wall, and the proliferation ability is strong.
2.BMSCs has the potential of multidifferentiation and can be induced to differentiate into osteoblasts and nerve like cells in vitro.
3., differentiated neuron like cells can be further differentiated into neurons and glial cells. They are ideal seed cells for stem cell transplantation, and have great potential for application.

【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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