人脐带间充质干细胞的生物学特性及神经分化研究

发布时间：2018-08-30 08:06

【摘要】：目的：研究人脐带间充质干细胞(MSCs)的生物学特性及丹参等药物诱导MSCs向神经细胞分化的可能性，为神经细胞移植、脐带组织库的建立及中药丹参作为神经诱导剂提供理论依据。方法：从人脐带华尔通胶(Wharton's jelly)分离培养MSCs，培养、检测人脐带来源的MSCs的细胞表面标记，评价其生物学特性；丹参和β—巯基已醇诱导人脐带来源的MSCs向神经细胞分化；用免疫细胞化学方法检测神经细胞相关蛋白标记的表达；用光镜、扫描电子显微镜监测丹参诱导后细胞形态改变；透射电子显微镜观察丹参诱导后细胞超微结构改变；用共聚焦显微镜分析丹参诱导前后细胞内DNA、RNA含量改变；用RT-PCR检测细胞神经相关基因pleiotrophin和神经干细胞的标记巢蛋白(nestin)的表达。结果：从人脐带华尔通胶分离、培养的贴壁细胞，体外生长形态类似于成纤维细胞，，可以维持在未分化状态稳定增殖，体外增殖超过10代，细胞冻存一个月后复苏，生长特点与冻存前基本一致。用流式细胞仪检测，这类细胞表达MSCs的表面标记CD29、CD44、CD59、CD90、CD105，不表达造血细胞表面标记CD14、CD33、CD34、CD28、CD45、CD117．和与移植免疫排斥相关的表面标记CD80(B7-1)、CD86(B7-2)、CD40。丹参和β—巯基已醇均可诱导人脐带MSCs向神经样细胞分化，分化的细胞表达神经干细胞的标记巢蛋白(nestin)，神经元的蛋白标记β-Ⅲ类神经微管(β-TubulinⅢ)和神经微丝(NF)，以及神
[Abstract]:Aim: to study the biological characteristics of human umbilical cord mesenchymal stem cells (MSCs) and the possibility of inducing MSCs to differentiate into nerve cells by drugs such as Salvia miltiorrhiza (Salvia miltiorrhiza), so as to provide theoretical basis for nerve cell transplantation, establishment of umbilical cord tissue bank and Chinese medicine Danshen as nerve inducer. Methods: MSCs, was isolated and cultured from human umbilical cord (Wharton's jelly), and the cell surface markers of MSCs derived from human umbilical cord were detected, and its biological characteristics were evaluated, and the differentiation of MSCs from human umbilical cord into neural cells was induced by Salvia miltiorrhiza and 尾 -mercaptohexanol. Immunocytochemistry was used to detect the expression of neuron-associated protein, light microscope and scanning electron microscope were used to monitor the morphological changes of cells induced by Salvia miltiorrhiza, and the ultrastructure of cells induced by Salvia miltiorrhiza was observed by transmission electron microscope. The changes of DNA,RNA content in cells before and after salvia miltiorrhiza induction were analyzed by confocal microscopy, and the expression of pleiotrophin and labeled nestin (nestin) in neural stem cells were detected by RT-PCR. Results: the adherent cells were isolated from human umbilical cord and cultured in vitro. The growth morphology of adherent cells was similar to that of fibroblasts. The cells could proliferate steadily in undifferentiated state and proliferate in vitro for more than 10 generations. The cells were resuscitated after one month of cryopreservation. The growth characteristics were basically consistent with those before freezing. By flow cytometry, CD29,CD44,CD59,CD90,CD105, a surface marker expressing MSCs, did not express hematopoietic cell surface marker CD14,CD33,CD34,CD28,CD45,CD117. and CD80 (B7-1) CD86 (B7-2) CD40, which was associated with allograft immune rejection. Salvia miltiorrhiza and 尾 -mercaptohexanol can induce human umbilical cord MSCs to differentiate into neuron-like cells. Differentiated cells express neural stem cells labeled with nestin (nestin), neurons labeled with 尾-鈪

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