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背景:体外培养扩增的骨髓间充质干细胞可诱导分化为神经细胞,但是有些诱导剂有一定的毒性,不能应用于人体内。目的:以黄芪为诱导剂,诱导大鼠骨髓间充质干细胞分化为神经样细胞。设计、时间及地点:随机对照实验,于2002-01/2005-01在大理学院基础医学院的云南省重点实验室和四川大学基础医学院与法医学院的干细胞研究中心完成。材料:6周龄健康SD大鼠,体质量120~130 g。方法:以密度梯度离心法从大鼠骨髓中分离培养骨髓间充质干细胞,并传代扩增。采含体积分数为0.125的黄芪无血清L-DMEM培养液诱导分化为神经样细胞,观察细胞形态变化,以免疫组织化学染色方法检测分化细胞中巢蛋白、神经元特异性烯醇化酶、胶质纤维酸性蛋白的表达。主要观察指标:①诱导分化细胞的免疫组织化学鉴定。②RT-PCR方法半定量分析相关基因Ngn-1和Wnt-1基因在诱导过程中的表达。结果:经传代后,骨髓间充质干细胞呈纤维细胞样,有较强的增殖能力。经黄芪诱导后,细胞形态发生改变,巢蛋白、神经元特异性烯醇化酶及胶质纤维酸性蛋白均阳性。Ngn-1和Wnt-1基因在黄芪诱导骨髓间充质干细胞过程中表达量升高。结论:骨髓间充质干细胞在黄芪诱导下可向神经样细胞分化。Ngn-1和Wnt-1基因在其分化过程中起正调控作用。
BACKGROUND: In vitro cultured bone marrow mesenchymal stem cells can be induced to differentiate into neural cells, but some inducers have certain toxicity and cannot be used in humans. OBJECTIVE: To induce the differentiation of rat bone marrow mesenchymal stem cells into neuron-like cells with astragalus as inducer. DESIGN, TIME AND SETTING: Randomized controlled trials were performed at the Yunnan Provincial Key Laboratory of the Basic Medical College of Dali University and the Stem Cell Research Center of the College of Basic Medicine and the School of Medicine of Sichuan University from January 2002 to January 2005. MATERIALS: Healthy 6-week-old SD rats, body weight 120-130 g. METHODS: Bone marrow mesenchymal stem cells were isolated and cultured from rat bone marrow by density gradient centrifugation and passaged for amplification. Different concentrations of Astragalus mongholicus serum-free L-DMEM containing 0.125 in volume were used to induce differentiation into neuron-like cells. Morphological changes were observed. Nestin, neuron-specific enolase, and glial were detected by immunohistochemical staining. Fibric acid protein expression. MAIN OUTCOME MEASURES: 1 Immunohistochemical identification of differentiated cells. 2RT-PCR method was used to semi-quantitatively analyze the expression of related genes Ngn-1 and Wnt-1 during the induction process. Results: After passage, bone marrow mesenchymal stem cells were fibroblast-like and had a strong ability to proliferate. After induction by Astragalus membranaceus, the cell morphology was changed, and nestin, neuron-specific enolase and glial fibrillary acidic protein were all positive. The expression of Ngn-1 and Wnt-1 genes increased during the induction of bone marrow mesenchymal stem cells by Astragalus membranaceus. CONCLUSION: Bone marrow mesenchymal stem cells can differentiate into neuron-like cells when induced by Astragalus membranaceus. Ngn-1 and Wnt-1 genes play a positive regulatory role in their differentiation.