中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (1): 41-46.doi: 10.3969/j.issn.2095-4344.1524

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

人脐带间充质干细胞向神经元样细胞诱导分化：两种方法的比较

王 武1，齐保军2，武忠炎1，崔 泳1   

  1. 1新疆医科大学第五附属医院骨一科，新疆维吾尔自治区乌鲁木齐市 830011；2开封市中心医院骨科，河南省开封市 475000
  • 修回日期:2018-09-05 出版日期:2019-01-08 发布日期:2018-11-28
  • 通讯作者: 崔泳，副主任医师，副教授，硕士生导师，新疆医科大学第五附属医院骨一科，新疆维吾尔自治区乌鲁木齐市 830011
  • 作者简介:王武，男，1982年生，陕西省西安市人，汉族，中南大学湘雅医院在读博士，主治医师，主要从事创伤骨科及软骨损伤的相关研究。 并列第一作者：齐保军，男，1984年生，黑龙江省海伦市人，汉族，2015年新疆医科大学毕业，硕士，医师，主要从事创伤骨科研究。
  • 基金资助:

    新疆维吾尔自治区自然科学基金项目(2016D01C239)，项目负责人：王武

Differentiation of human umbilical cord mesenchymal stem cells into neuron-like cells: a comparison of two induction methods

Wang Wu1, Qi Baojun2, Wu Zhongyan1, Cui Yong1   

  1. 1First Department Orthopedics, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China; 2Department of Orthopedics, Kaifeng Central Hospital, Kaifeng 475000, Henan Province, China
  • Revised:2018-09-05 Online:2019-01-08 Published:2018-11-28
  • Contact: Cui Yong, Associate chief physician, Associate professor, Master’s supervisor, First Department Orthopedics, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
  • About author:Wang Wu, Doctoral candidate, Attending physician, First Department Orthopedics, the Fifth Affiliated Hospital of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China. Qi Baojun, Master, Physician, Department of Orthopedics, Kaifeng Central Hospital, Kaifeng 475000, Henan Province, China. Wang Wu and Qi Baojun contributed equally to this work.
  • Supported by:

    the Natural Science Foundation of Xinjiang Uygur Autonomous Region, No. 2016D01C239 (to WW)

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摘要：

文章快速阅读：

文题释义：
脐带间充质干细胞：是存在于新生儿脐带组织中的一种多能干细胞，具有强大的增殖能力和多向分化潜能；其具有来源丰富、免疫原性低、移植后不需应用免疫抑制剂可以长期存活等诸多特性，在临床应用中具有广阔的前景。
干细胞向神经元样细胞诱导分化：干细胞具有多向分化潜能，其在诱导剂(2% B27、20 μg/L碱性成纤维细胞生长因子、20 μg/L表皮生长因子)的作用下可进行一系列分裂和分化，并能发育成为成熟的神经元样细胞；分化后的细胞可表现出神经细胞的形态特征(细胞呈星形，部分细胞胞质收缩形成突起，细胞胞体形成多样的神经样轴突，细胞之间形成网络样连接)，并能表达神经细胞特异性标志物。

 

摘要
背景：干细胞因其可向神经元样细胞诱导分化而在临床神经损伤疾病治疗中具有广阔的前景，但干细胞向神经元样细胞诱导分化的效率不高，为此作者对传统的诱导方法进行了改良。
目的：探讨2种不同方法诱导人脐带间充质干细胞向神经元样细胞分化的潜能及2种诱导方法的优劣。
方法：采用组织块法分离培养人脐带间充质干细胞，显微镜下观察其形态并利用流式细胞仪进行干细胞鉴定，在神经营养因子诱导方案下，采用相同的诱导剂(2% B27、20 μg/L碱性成纤维细胞生长因子、20 μg/L表皮生长因子)，2种不同的诱导方法将其向神经元样细胞诱导分化(传统方法：将B27、碱性成纤维细胞生长因子、表皮生长因子及青链霉素合剂加入DMEM/F12培养基中进行诱导，3 d后加入全反式维甲酸，共培养10 d；改良方法：将B27、碱性成纤维细胞生长因子、表皮生长因子、青链霉素合剂及胎牛血清加入DMEM/F12培养基中进行诱导，6 d后用胰酶消化后重新接种于共聚焦小皿内，次日去除B27和胎牛血清，并加入全反式维甲酸，共培养14 d)。应用免疫荧光染色对诱导分化后细胞表面神经标志物——神经丝蛋白(NF-M)和胶质纤维酸性蛋白(GFAP)进行检测，比较2种方法诱导后细胞表面神经标志物的阳性表达率。
结果与结论：①成功分离出人脐带间充质干细胞，经流式细胞仪鉴定其高表达CD29、CD105，低表达CD34、CD45，符合干细胞特性；②2种方法诱导后，镜下观察到的细胞均可呈现神经细胞样改变，免疫荧光检测提示2种方法诱导后细胞中神经丝蛋白与胶质纤维酸性蛋白均呈阳性表达；③传统方法组神经丝蛋白与胶质纤维酸性蛋白的阳性率分别为18.73%和18.01%，改良方法组二者的阳性率分别为27.48%和29.24%，两组比较差异有显著性意义(P < 0.05)；④结果表明，2种诱导方法均可将人脐带间充质干细胞诱导分化为神经元样细胞；但在诱导分化后的细胞形态、神经标志物(神经丝蛋白与胶质纤维酸性蛋白)的阳性表达率方面，改良方法更具有优势。

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0001-9791-9491(王武)

关键词: 人脐带间充质干细胞, 诱导分化, 神经细胞, 神经丝蛋白, 胶质纤维酸性蛋白, 干细胞

Abstract:

BACKGROUND: Stem cells are a promising treatment for nerve injury diseases due to its ability of differentiating into neuron-like cells. However, the differentiation efficacy is low, so we have make an advance based on the traditional induction method.
OBJECTIVE: To explore the potentiality of human umbilical cord mesenchymal stem cells differentiating into neuron-like cells induced by two methods and their merits.
METHODS: Human umbilical cord mesenchymal stem cells were isolated and cultured using tissue explant method. The cell morphology was observed under microscope, and the cultured cells were identified by flow cytometry. Human umbilical cord mesenchymal stem cells were differentiated into neuron-like cell under the neurotrophic factor inducing scheme. Two different methods but using the same inducers (2% B27, 20 μg/L basic fibroblast growth factor and 20 μg/L epidermal growth factor) were utilized. Traditional method: B27, basic fibroblast growth factor, epidermal growth factor and penicillin streptomycin combination were added in the DMEM/F12 medium, 3 days later all-trans retinoic acid was added, followed by 10 days of culture. Advanced method: B27, basic fibroblast growth factor, epidermal growth factor, penicillin streptomycin combination and fetal bovine serum were added in the DMEM/F12 medium, 6 days later the cells underwent trypsinization and re-seeded in the confocal capsule, B27 and fetal bovine serum were removed next day, and all-trans retinoic acid was added, cultured for 14 days. Then the differentiated nerve cells were identified by immunofluorescence detection of neurofilament protein and glial fibrillary acidic protein. Meanwhile, the positive rate of the neuromarkers in the cells was compared between two methods.
RESULTS AND CONCLUSION: Primary human umbilical cord mesenchymal stem cells were isolated and cultured successfully, and highly expressed CD29 and CD105 whereas lowly expressed CD34 and CD45, which were the characteristics of the stem cell. The differentiated cells were presented as nerve cells in the morphology under microscope in both inducing methods. The results of the immunofluorescence indicated that the neurofilament protein and the glial fiber acidic protein in the differentiated cells were all expressed positive in both methods. The positive rates of neurofilament protein and glial fiber acidic protein were 18.73% and 18.01% for the traditional method, while 27.48% and 29.24% for the advanced methods, respectively (P < 0.05). In summary, human umbilical cord mesenchymal stem cells can differentiate into the neuron-like cells induced by two methods. Moreover, the advanced method is better than the traditional method in the morphology of the differentiated cells and the positive rate of neurofilament protein and glial fiber acidic protein. 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Cell Differentiation, Neurons, Tissue Engineering

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