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背景:近年来通过筛选不同细胞因子的诱导分化作用发现,某些特定的细胞因子配伍可明显诱导中脑神经干细胞体外定向分化成多巴胺能神经元,研究还发现神经干细胞体外诱导分化的多巴胺能神经元可以有效应用于移植治疗帕金森病,为提高其体内移植的疗效,目前迫切需要深入研究神经干细胞诱导分化的生物学特性。目的:探讨大鼠神经干细胞在分化液中诱导不同时间后,其体外分化成多巴胺能神经元的效应。设计:单一样本观察。单位:中山大学附属第一医院神经外科。材料:实验于2007-05/10在中山大学附属第一医院完成。选择清洁级孕14d的健康SD大鼠6只,体质量350~400g,由中山大学动物实验中心提供(许可证号码SCXK(粤)2007-0034)。实验过程中对动物处置符合动物伦理学标准。方法:①在含表皮生长因子及碱性成纤维细胞生长因子的无血清培养液中培养胚胎大鼠中脑神经干细胞。②经传代扩增后,在含白细胞介素1、白细胞介素11、白血病抑制因子、胶质细胞源性神经营养因子的诱导分化液中向多巴胺能神经元分化。③诱导分化2,4,6,8,10d后,流式细胞仪检测分化细胞中酪氨酸羟化酶阳性细胞的比值。主要观察指标:①大鼠神经干细胞诱导分化后细胞形态的变化。②诱导不同时间的大鼠神经干细胞分化成酪氨酸羟化酶阳性细胞的比值。结果:在诱导分化液中大鼠中脑神经干细胞球呈贴壁生长,球形结构开始塌陷,球内细胞从球体中央逐渐向四周分化扩展出较多形态各异的细胞,分化6d后,多数细胞已生长出一两个长突起及多个短突起。免疫细胞化学显示分化的细胞中含有酪氨酸羟化酶染色阳性细胞,流式细胞仪检测诱导分化2,4,6,8,10d的分化细胞中酪氨酸羟化酶染色阳性细胞的比例分别为(3.2±0.9)%,(6.8±1.6)%,(16.7±2.6)%,(14.8±1.8)%,(12.2±2.5)%,各组间差异有显著性意义(F=26.449,P<0.05)。结论:诱导时间对大鼠中脑神经干细胞体外分化成多巴胺能神经元的能力存在影响,诱导6d的神经干细胞分化成多巴胺能神经元的比例最高。
BACKGROUND: In recent years, through screening and differentiation of different cytokines, it was found that certain specific cytokines can obviously induce the differentiation of mesencephalic neural stem cells into dopaminergic neurons in vitro. The study also found that neural stem cells induced differentiated dopaminergic neurons in vitro Yuan can be effectively used in the treatment of Parkinson's disease. In order to improve the efficacy of transplantation in vivo, there is an urgent need to further study the biological characteristics of neural stem cell-induced differentiation. AIM: To investigate the effect of differentiating neural stem cells into dopaminergic neurons in vitro after differentiation of rat neural stem cells for different periods of time. Design: Single sample observation. Unit: Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University. Materials: The experiment was performed at the First Affiliated Hospital of Sun Yat-sen University from May to October 2007. Six healthy SD rats of cleaning grade 14d were selected and their body weight was 350-400g. They were provided by Animal Experiment Center of Sun Yat-sen University (license No. SCXK 2007-0034). Animal experiments in the process of animal ethical standards. Methods: ① Embryonic rat middle cerebral neural stem cells were cultured in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. (2) After passage, the cells differentiated into dopaminergic neurons in induced differentiation medium containing IL-1, IL-11, leukemia inhibitory factor and glial cell line-derived neurotrophic factor. ③ After differentiation for 2, 4, 6, 8 and 10 days, the ratio of tyrosine hydroxylase positive cells in differentiated cells was detected by flow cytometry. MAIN OUTCOME MEASURES: ① The morphological changes of rat neural stem cells after differentiation. ② inducing rat neural stem cells differentiated into tyrosine hydroxylase-positive cells at different times. Results: In the induced differentiation medium, the NSCs spheroids grew adherently and the globular structures began to collapse. The globular cells gradually differentiated from the center to the periphery of the sphere and expanded more cells with different morphology. After 6 days of differentiation, most of the cells One or two long protrusions and several short protrusions have been grown. Immunocytochemistry showed that the differentiated cells contained tyrosine hydroxylase staining positive cells, and the proportion of tyrosine hydroxylase staining positive cells in differentiation cells induced by differentiation for 2, 4, 6, 8, and 10 d was detected by flow cytometry The difference was statistically significant (F = 26.449, P <0.05), and the difference was significant (P <0.05) P <0.05). CONCLUSION: The induction time has an effect on the ability of rat neural stem cells to differentiate into dopaminergic neurons in vitro. The proportion of neural stem cells that differentiate into dopaminergic neurons on day 6 is the highest.