摘要
目的探讨骨髓间充质干细胞体外诱导成为黑素细胞的可能性。方法6周龄雄性C57BL/6小鼠股骨基质细胞行原代培养,6次传代后以氢化可的松、重组人胰岛素、转铁蛋白和成纤维细胞生长因子诱导黑素细胞。倒置光学显微镜观察细胞分化;透射电镜观察黑素小体成熟;免疫荧光染色观察黑素细胞相关表位表达;流式细胞仪检测黑素细胞的细胞周期及获得率。结果6次传代间充质干细胞数量近109,免疫荧光检测CD44阳性率94.3%和CDl05阳性率82.3%。培养180d,细胞形态接近于黑素细胞,树突增多,胞质内出现黑素小体样结构,生长周期加快为3~4d,肉眼可见棕黑色细胞沉淀。电镜观察显示Ⅳ期为主的黑素小体。免疫荧光显示酪氨酸酶相关蛋白-1,酪氨酸酶相关蛋白-2和小眼畸形相关转录因子阳性。流式细胞仪分析显示细胞基本处于G,和S期。酪氨酸酶相关蛋白-1阳性的黑素细胞获得率约为80%。结论骨髓间充质干细胞可以被大量诱导分化为黑素细胞;诱导黑素细胞的形态学、超微结构、特异性表位等皆接近于正常黑素细胞.具有一定的增殖活性.获得率较高。
Objective To explore the possibility of bone marrow mesenchymal stem cells (MSCs) differentiating into active melanocytes in vitro. Methods Bone marrow stromal cells were harvested from femoral marrow of 6-week-old black male C57BL/6 mice, and subjected to a primary culture. After 6-passage subculture, an induction medium containing hydrocortisone, recombinant human insulin, transferrin and fibroblast growth factor was used to induce the differentiation of MSCs into melanocytes. Inverted light microscopy was applied to observe the process of cell differentiation, transmission electron microscopy to observe melanosome formation and maturation, and immunocytochemistry to determine the expression of melanocyte-associated epitopes, and flow cytometry to analyze cell cycles and yield of differentiated melanoeytes. Results The total number of MSCs was close to 109 after 6 passages of subculture, and immunofluorescent studies showed an expression rate of 94.3% for CD44 and 82.3% for CD105 in these MSCs. After 180-day cultivation in the induction medium, the MSCs showed a morphological similarity to melanocytes with an increase in dendrites, formation of melanosome-like structures, and cell growth cycle was shortened to 3 - 4 days. Brown/black cell sediments were visualized by naked eyes. Electron microscopy revealed that intraeellular melanosomes were mainly in IV phase. Immunofluorescence studies of the differentiated melanoeytes showed a positive staining for tyrosinase related protein-1 (TRP-1), TRP-2, and microphthalmia-assoeiated transcription factor (MITF). Flow eytometric analysis showed that most of the melanoeytes differentiated from the MSCs were in G1 and S phases, and TRP-l-positive melanocytes amounted to 80% of gate cells. Conclusions Bone marrow MSCs can be largely differentiated into melanoeytes with a close similarity to normal melanocytes in morphology, ultrastructure and specific epitopes and a certain degree of proliferative activity.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2013年第5期336-340,共5页
Chinese Journal of Dermatology
基金
上海市科委重点攻关项目(11DZl973000),上海市卫生局项目(2010J006A)
关键词
间质干细胞
骨髓
黑素细胞
体外研究
Mesenchymal stem cells
Bone marrow
Melanocytes
In vitro