中华皮肤科杂志 ›› 2014, Vol. 47 ›› Issue (2): 85-89.

• 论著 • 上一篇    下一篇

人脂肪间充质干细胞体外向黑素细胞分化的研究

闵敏1,张雪静2,马红3,许辉4,李遇梅3   

  1. 1. 浙江大学医学院附属第二医院
    2. 江苏省镇江市第二人民医院
    3. 江苏大学附属医院
    4. 镇江江苏大学附属医院皮肤科
  • 收稿日期:2013-01-04 修回日期:2013-10-19 出版日期:2014-02-15 发布日期:2014-02-01
  • 通讯作者: 李遇梅 E-mail:liyumeimg2000@yahoo.com.cn
  • 基金资助:
    国家自然科学基金;中华医学会皮肤性病学分会－安斯泰来皮肤病学研究基金

In vitro induction of differentiation of human adipose-derived mesenchymal stem cells into melanocytes: an experimental study

  • Received:2013-01-04 Revised:2013-10-19 Online:2014-02-15 Published:2014-02-01
  • Contact: LI Yu-Mei E-mail:liyumeimg2000@yahoo.com.cn

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摘要： 【摘要】 目的 建立体外诱导人脂肪间充质干细胞（hADSC）向黑素细胞分化的方法。 方法 由人皮下脂肪分离培养获得hADSC，流式细胞仪检测细胞表型，成骨成脂分化证明分化潜能。将干细胞生长因子（SCF）、内皮素3 （EDN-3）、碱性成纤维细胞生长因子（bFGF）等加到条件培养基中，促使第5代hADSC向黑素细胞方向诱导，诱导至第10周。未诱导组为正常对照组。使用实时荧光定量PCR检测黑素相关基因小眼畸形转录因子（MITF），酪氨酸激酶受体c-Kit（KIT），多巴色素异构酶（DCT），酪氨酸酶（TYR），酪氨酸酶相关蛋白1（TYRP1），性别决定区域相关转录因子10（SOX10） mRNA的表达量，统计学分析采用单因素方差分析及LSD-t检验。诱导结束后，通过免疫细胞化学法、细胞免疫荧光法进行鉴定。 结果 流式细胞仪显示分离培养获得的hADSC高表达CD29、CD44、CD73、CD90、CD105、CD166，阳性率均在95%以上；低表达或不表达CD31、CD34、CD45，人白细胞DR抗原（HLA-DR）。成骨成脂分化结果显示hADSC具有分化潜能。实时荧光定量PCR结果显示，诱导10周后，MITF、KIT、DCT、TYR、TYRP1、SOX10 mRNA的表达水平分别为0.325 ± 0.012，0.042 ± 0.006，0.046 ± 0.013，0.036 ± 0.005，0.041 ± 0.003，0.225 ± 0.014，与诱导0周组相比，均有上调（P < 0.05）。诱导结束后，免疫细胞化学MITF、HMB45染色阳性，细胞免疫荧光TYRP1、S100阳性表达。 结论 由人脂肪间充质干细胞诱导所得细胞具有一定的黑素细胞生物学特性。

关键词: 人脂肪间充质干细胞, 分化, 黑素细胞

Abstract: Min Min*, Zhang Xuejing, Ma Hong, Xu Hui, Li Yumei. *Department of Dermatology, Affiliated Hospital of Jiangsu University, Zhenjiang 212000, Jiangsu, China Corresponding author: Li Yumei, Email: l.yumei@aliyun.com 【Abstract】 Objective To establish a method for inducing human adipose-derived mesenchymal stem cells （hADSCs） to differentiate into melanocytes in vitro. Methods hADSCs were isolated from human subcutaneous fat tissue, and subjected to phenotypic analysis by flow cytometry. The differentiation potential of the hADSCs was evaluated by induction of osteogenic and adipogenic differentiation. Then, the fifth passage hADSCs were induced to differentiate into melanocytes by conditioned medium containing stem cell factor （SCF）, endothelin 3, basic fibroblast growth factor （bFGF） for ten weeks. Those hADSCs receiving no induction treatment served as the normal control. Subsequently, real-time fluorescence-based quantitative PCR （RT-PCR） was performed to determine the mRNA expressions of melanin-associated genes including microophthalmia-associated transcription factor （MITF）, c-kit receptor tyrosine kinase （KIT）, dopachrome tautomerase （DCT）, tyrosinase, tyrosinase-related protein-1 （TYRP1）, and sex determining region Y- box 10 （SOX10）. Statistical analysis was done by one-factor analysis of variance and the least significant difference （LSD） test. Further more, the hADSC-derived melanocytes were identified by immunocytochemistry and immunofluorescence assay. Results As flow cytometry revealed, the isolated hADSCs showed high expressions of CD29, CD44, CD73, CD90, CD105 and CD166 with the positivity rates being more than 95%, but low or absent expressions of CD31, CD34, CD45 and human leukocyte antigen （HLA）-DR. Additionally, the osteogenic and adipogenic differentiation of hADSCs was successfully induced. The relative mRNA expression levels of MITF, KIT, DCT, tyrosinase，TYRP1 and SOX10 were 0.325 ± 0.012, 0.042 ± 0.006, 0.046 ± 0.013, 0.036 ± 0.005, 0.041 ± 0.003, and 0.225 ± 0.014 respectively in hADSCs induced for 10 weeks，which were significantly upregulated compared with the normal control hADSCs （all P < 0.05）. Moreover, the induced hADSCs showed positive immunocytochemical staining for MITF and HMB45, as well as positive cytoimmunofluorescence staining for TYRP1 and S100. Conclusion The hADSCs have been successfully induced to differentiate into cells with the biological characteristics of melanocytes.

Key words: Human adipose-derived mesenchymal stem cells, Cell differentiation, Melanocytes

中图分类号: 

  • R751.05