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背景:研究发现骨髓间质干细胞在体外专门的诱导体系中能产生类似神经元烯醇化酶克隆球样神经球结构,故骨髓间质干细胞成为中枢神经损伤修复种子细胞的热门候选。目的:观察体外分化体系诱导大鼠骨髓间质干细胞分化为神经元样细胞的情况。设计:观察性实验。单位:深圳市第二人民医院脊柱外科。材料:选用孕16d及2月龄的SD大鼠,由华中科大同济医学院动物中心提供。神经元烯醇化酶单抗、胶质纤维酸性蛋白多抗、神经中丝蛋白单抗均购自武汉博士德公司。方法:实验于2006-09在华中科技大学同济医学院基础医学部免疫室开放实验室及本院中心实验室完成。取大鼠下肢骨,离心分离鼠骨髓间质干细胞。提取孕16d大鼠胚胎脊髓组织匀浆,制备诱导液,诱导骨髓间质干细胞的分化。另取胚胎肌组织同法制备肌组织匀浆作对照。主要观察指标:对诱导后骨髓间质干细胞进行形态学观察及用抗神经元烯醇化酶、NF200、抗胶质纤维酸性蛋白分别标记神经元,星形胶质细胞。反应阳性诱导细胞免疫组化染色后光镜下随机数取10个非重叠视野进行观察,计算神经元烯醇化酶和神经中丝蛋白阳性细胞占总细胞数的比例。结果:①大鼠骨髓间质干细胞在诱导后细胞形态变化:诱导早期光镜下即见部分细胞胞体回缩,突起变长,变细;逐渐出现分化细胞突起生长,并相互交织,类似神经细胞,有的分支类似树突状分枝,对照组细胞形态变化不大。②诱导1周后,骨髓基质干细胞向神经元样细胞分化相关抗原表达情况:脊髓匀浆组多数细胞表现为神经元烯醇化酶和神经中丝蛋白阳性;少数细胞表达胶质纤维酸性蛋白,对照组无神经细胞抗体阳性染色出现。而对照组未见神经细胞抗原反应阳性。免疫组化发现分化细胞神经元烯醇化酶、神经中丝蛋白表达阳性率分别为(68±1.7)%,(76.2±2.9)%。结论:胚胎脊髓组织匀浆可诱导骨髓间质干细胞分化为神经元样细胞。
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) can generate neuron-like structures similar to neuron enolase in specialized in vitro induction system. Therefore, bone marrow mesenchymal stem cells (BMSCs) become hot candidates for repairing seed cells of central nervous system. Objective: To observe the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells induced by in vitro differentiation system. Design: Observational experiments. Unit: Second People 's Hospital of Shenzhen City Spinal Surgery. MATERIALS: SD rats of 16d and 2 months pregnant were selected and provided by Animal Center of Tongji Medical College of Huazhong University of Science and Technology. Neuron enolase monoclonal antibody, glial fibrillary acidic protein polyclonal antibody, nerve silk protein monoclonal antibody were purchased from Wuhan Boster company. Methods: The experiment was performed at the Laboratory of Immunization Room and Laboratory of Tongji Medical College of Tongji Medical College, Huazhong University of Science and Technology from September 2006 to September 2006. Rat lower extremity bone was taken and centrifuged to separate rat bone marrow mesenchymal stem cells. The embryo spinal cord tissue homogenate was extracted from the pregnant 16-day-old rats to prepare the inducing solution to induce the differentiation of bone marrow mesenchymal stem cells. Another fetus muscle tissue preparation method with the law as control. MAIN OUTCOME MEASURES: Bone marrow mesenchymal stem cells were observed for morphology and neurons and astrocytes were labeled with anti-neuron enolase, NF200, anti-glial fibrillary acidic protein. After reaction-positive cells were stained with immunohistochemical method, 10 non-overlapped fields were observed under light microscope. The proportion of neuron enolase and neurofilament positive cells in total cells was calculated. Results: (1) The morphological changes of rat bone marrow mesenchymal stem cells after induction: early part of the cell body cells retracted under the light microscope, the protrusions became longer and thinner, and the differentiated cells gradually grew and intertwined, similar to nerve cells , Some branches similar to dendritic branches, the control group cells did not change much. ② After 1 week of induction, the expression of bone marrow stromal stem cells differentiate into neuron-like cells: Most of the cells in the spinal cord homogenate group showed positive neuron enolase and neurofilament protein; few cells expressed glial fibrillary acidic protein, Group no neuronal antibody positive staining appeared. The control group showed no neuronal antigen-positive. The positive rates of neurofilament and neurofilament expression in differentiated cells were (68 ± 1.7)% and (76.2 ± 2.9)%, respectively. Conclusion: Bone marrow mesenchymal stem cells can be induced to differentiate into neuron - like cells by embryonic spinal cord homogenate.