中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (50): 7500-7506.doi: 10.3969/j.issn.2095-4344.2016.50.008

• 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分化胰岛样细胞过程中胰岛素和巢蛋白的表达

申  义1，王意忠1，时  瀚1，山  霞1，王琳琳1，崔晓兰2   

  1. 1北京大学航天临床医学院血液内分泌科，北京市  100049
    2中国中医科学院中药所药理室，北京市  100700
  • 修回日期:2016-09-18 出版日期:2016-12-02 发布日期:2016-12-02
  • 通讯作者: 王意忠，博士，北京大学航天临床医学院血液内分泌科，北京市100049
  • 作者简介:申义，男，1987年生，河北省邯郸市人，汉族，在读硕士，主要从事干细胞治疗的基础研究。
  • 基金资助:

    航天中心医院科研基金(KY-201003)，课题名称：人骨髓间充质干细胞向胰岛细胞定向分化的研究

Changes of insulin and nestin expression during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells

Shen Yi1, Wang Yi-zhong1, Shi Han1, Shan Xia1, Wang Lin-lin1, Cui Xiao-lan2   

  1. 1Department of Hematology and Endocrinology, Peking University Aerospace School of Clinical Medicine, Beijing 100049, China
    2Pharmacology Laboratory, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700,China
  • Revised:2016-09-18 Online:2016-12-02 Published:2016-12-02
  • Contact: Wang Yi-zhong, M.D., Department of Hematology and Endocrinology, Peking University Aerospace School of Clinical Medicine, Beijing 100049, China
  • About author:Shen Yi, Studying for master’s degree, Department of Hematology and Endocrinology, Peking University Aerospace School of Clinical Medicine, Beijing 100049, China
  • Supported by:

    the Scientific Research Foundation of Peking University Aerospace School of Clinical Medicine in China, No. KY-201003

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摘要：

文章快速阅读：

文题释义：
巢蛋白：是神经干细胞、胰腺干细胞的标志分子，被列为第Ⅵ类中间纤维，其结构和分子质量与其他的中间纤维成员有差异。神经干细胞和胰岛干细胞在胚胎发育早期都处于特征性的后有丝分裂，表达许多相同的表型，并且都可以分泌巢蛋白，在体外一定条件下，巢蛋白阳性细胞能够定向分化为胰腺的内、外分泌细胞。
双硫腙：俗称铅试剂，化学试剂。蓝黑色结晶性粉末，易被空气氧化，可加入二氧化硫水溶液保护；易溶于四氯化碳和氯仿，其溶液不稳定，微溶于乙醇，不溶于水。胰岛细胞胞浆内含有高浓度锌离子，其可促进形成2-锌-胰岛素六聚体，而后者可以调节人体血糖水平，因此检测细胞中锌离子水平对鉴定胰岛细胞具有重要价值。双硫腙能特异性的和锌离子螯合成棕红色络合物，使胰岛细胞显色，其他细胞不显色。

 

摘要
背景：国内外研究证实脐带间充质干细胞可定向诱导分化为胰岛样细胞，但分化过程中胰岛素与巢蛋白表达的变化等相关研究甚少。
目的：观察人脐带间充质干细胞向胰岛素样细胞分化过程中胰岛素与巢蛋白表达的变化。
方法：采用UltraCULTURE培养基在体外培养人脐带间充质干细胞，取培养的第3代细胞，按照两阶段诱导方案将其定向诱导分化为胰岛样细胞，第1阶段：在UltraCULTURE培养基中加入4 nmol/L 活化素A，25 μg/L表皮生长因子，100 μg/L β-神经生长因子，10 mmol/L尼克酰胺，诱导培养至14 d。第2阶段：在UltraCULTURE培养基中加入1%胰岛素-转铁蛋白-硒，10 mmol/L尼克酰胺，10 μg/L碱性成纤维细胞生长因子，继续诱导培养14 d。在诱导分化过程中，采用流式细胞技术检测分化细胞巢蛋白及胰岛素的表达，双硫腙染色鉴定胰岛样细胞团中锌离子的表达。
结果与结论：①诱导分化为胰岛样细胞过程中胰岛素水平逐渐增高，至28 d达到较高水平，未诱导组胰岛素表达呈阴性；②诱导分化第14天，巢蛋白表达水平达到最大，随着诱导时间延长表达水平逐渐下降；③脐带间充质干细胞诱导28 d形成胰岛样细胞团，双硫腙染色阳性；④实验将脐带间充质干细胞成功诱导分化为胰岛样细胞，并且随着定向分化细胞的变化，细胞胰岛素与巢蛋白表达水平也出现相应的变化。

 

 

关键词: 干细胞, 脐带脐血干细胞, 脐带间充质干细胞, 胰岛样细胞, 诱导分化, 胰岛素, 巢蛋白

Abstract:

BACKGROUND: Domestic and international studies have confirmed that human umbilical cord mesenchymal stem cells could be induced to differentiate into islet-like cells, but little is reported about the changes of insulin and nestin expressions during the differentiation phase.
OBJECTIVE: To observe the changes of insulin and nestin expressions during the differentiation of human umbilical cord mesenchymal stem cells into islet-like cells.
METHODS: Human umbilical cord mesenchymal stem cells were cultured using UltraCULTURE medium in vitro. Stem cells were cultured for three generations to observe cell morphological changes under an inverted microscope, to test immunophenotype by flow cytometry, and to identify the capacity of osteogenesis and adipogenic differentiation. Induction protocol was divided into two stages. In stage 1, stem cells were induced for 14 days in the UltraCULTURE medium with 4 nmol/L activin A, 25 μg/L epidermal growth factor, 100 μg/L β-nerve growth factor, 10 mmol/L nicotinamide. In stage 2, the cells were cultured in the UltraCULTURE medium with 1% insulin-transferin-selenium, 10 mmol/L nicotinamide, 10 μg/L basic fibroblast growth factor for an additional 14 days. The expressions of nestin and insulin in those differentiated cells were tested by flow cytometry, and zinc ion expression in the islet-like cell clusters was identified by dithizone staining.
RESULTS AND CONCLUSION: During the differentiation process, the insulin level was increased gradually in the induction group and reached a higher level on day 28, but the insulin expression showed negative in the control group. In addition, on day 14 of induced differentiation, the nestin expression reached the peak and then gradually reduced along with the prolonged inductive time. On day 28 of induction, islet-like cell clusters formed and were positive for dithizone staining. In this experiment, the umbilical cord mesenchymal stem cells were successfully induced and differentiated into islet-like cells, accompanied with the variation of insulin and nestin expression.

 

 

Key words: Umbilical Cord, Mesenchymal Stem Cells, Insulin-Secreting Cells, Insulin, Tissue Engineering

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