体外诱导小鼠骨髓间充质干细胞分化为黑素细胞的观察

摘要/Abstract

摘要： 【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CD105阳性率82.3%。培养180 d，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 ~ 4 d，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G1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。【关键词】间质干细胞；骨髓；黑素细胞；体外研究

关键词: 黑素细胞, 间质干细胞, 骨髓, 体外研究

Abstract: SHI Wei-min, ZHU Jian-yu, SUN Yue, ZHU Guang-dou. Department of Dermatology, First People's Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200080, China Corresponding author: SHI Wei-min, Email: shiweimin1234@medmail.com.cn 【Abstract】 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 melanocytes. 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 intracellular melanosomes were mainly in IV phase. Immunofluorescence studies of the differentiated melanocytes showed a positive staining for tyrosinase related protein-1 （TRP-1）, TRP-2, and microphthalmia-associated transcription factor （MITF）. Flow cytometric analysis showed that most of the melanocytes differentiated from the MSCs were in G1 and S phases, and TRP-1-positive melanocytes amounted to 80% of gate cells. Conclusions Bone marrow MSCs can be largely differentiated into melanocytes with a close similarity to normal melanocytes in morphology, ultrastructure and specific epitopes and a certain degree of proliferative activity. 【Key words】 Mesenchymal stem cells; Bone marrow; Melanocytes; In vitro

Key words: Melanocyte, Mesenchymal stem cells, Bone marrow

施伟民朱建宇孙越朱光斗. 体外诱导小鼠骨髓间充质干细胞分化为黑素细胞的观察[J]. 中华皮肤科杂志, 2013, 46(5): 336-340.

参考文献

[1] Patel NS, Paghdal KV, Cohen GF. Advanced treatment modalities for vitiligo. Dermatol Surg. 2012, 38(3):381-391. [2] Cimadamore F, Shah M, Amador-Arjona A, et al.SOX2 Modulates Levels of MITF in Human Embryonic Stem Cell - Derived Neural Crest, Normal Human Melanocytes, and Melanoma Lines In Vitro. Pigment Cell Melanoma Res. 2012 May 9. [Epub ahead of print] [3] Yang R, Jiang M, Kumar SM, et al. Generation of melanocytes from induced pluripotent stem cells. J Invest Dermatol. 2011, 131(12):2458-2466. [4] Tirino V, Paino F, De Rosa A, et al. Identification, isolation, characterization, and banking of human dental pulp stem cells. Methods Mol Biol. 2012, 879:443-463. [5] Paino F, Ricci G, De Rosa A, et al. Ecto-mesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes. Eur Cell Mater. 2010, 20:295-305. [6] Yew TL, Chang MC, Hsu YT, et al. Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions. J Tissue Eng Regen Med. 2012 May 24. [Epub ahead of print] [7] 刘卫东, 任彩萍, 姚开泰. 人胚胎干细胞的体外定向分化. 生命科学, 2007, 19(5):518-525. [8] Tirino V, Paino F, d'Aquino R, et al. Methods for the identification, characterization and banking of human DPSCs: current strategies and perspectives. Stem Cell Rev. 2011; 7(3): 608-615. [9] Alikhan A, Felsten LM, Daly M, et al. Vitiligo: a comprehensive overview Part I. Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, histopathology, etiology, and work-up. J Am Acad Dermatol. 2011, 65(3):473-91. [10] Patel NS, Paghdal KV, Cohen GF. Advanced treatment modalities for vitiligo. Dermatol Surg. 2012, 38(3):381-391. [11] Saitoh A, Kawanabe T, Weidong H,et al. Selective upregulation of fibroblast Fas ligand expression, and prolongation of Fas/Fas ligand-mediated skin allograft survival, by retinoic acid: the skin as a retinoide-inducible immune privilege site. J Invest Dermatol. 2000, 115(2): 154-161. [12] 董达科, 项蕾红. 毛囊干细胞向黑素细胞系定向分化调控机制的研究进展. 国际皮肤性病学杂志. 2011, 37(4): 255-257. [13] Li Y, Chen J, Wang L,et al. Intracerebral transplantation of bone marrow stromal cells in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Neurosci Lett. 2001, 316(2):67-70. [14] Malgieri A, Kantzari E, Patrizi MP, et al. Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. Int J Clin Exp Med. 2010, 3(4):248-269. [15] Ke-Xin S, Qun Q, Da-Qing L, et al. Application of melanocytes and bone marrow mesenchymal stem cells in tissue engineered skin construction. Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2011, 33(4):402-407. [16] Kagami H, Agata H, Tojo A, et al. Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. Int J Biochem Cell Biol. 2011, 43(3):286-289. [17] Halaban R. Signal transduction in normal and malignant melanocytes. Pigment Cell Res. 1994, 7(2):89-95. [18] Murisier F, Beermann F. Genetics of pigment cells: lessons from the tyrosinase gene family. Histol Histopathol. 2006, 21(5):567-578. [19] Aksan I, Goding CR. Targeting the microphthalmia basic helix-loop-helix-leucine zipper transcription factor to a subset of E-box elements in vitro and in vivo. Mol Cell Biol. 1998,18(12):6930-6938. [20] Levy C, Khaled M, Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med. 2006,12: 406-414.