中华皮肤科杂志 ›› 2009, Vol. 42 ›› Issue (1): 49-51.

• 论著 • 上一篇    下一篇

人表皮成黑素细胞培养条件的初步探讨

赵志国 丁克云 金城 陆洪光 尹雪锋   

  1. 镇江市第一人民医院 (江苏大学附属人民医院)皮肤科 江苏省镇江市江苏大学附属镇江市第一人民医院皮肤科 南京医科大学附属无锡第二医院皮肤科 贵州贵阳医学院附属医院
  • 收稿日期:2008-03-06 修回日期:2008-03-28 出版日期:2009-01-15 发布日期:2009-01-15
  • 通讯作者: 赵志国 E-mail:pfzzg5665ok@sina.com

Investigations into the culture condition for human epidermal melanoblasts in vitro

lu hongguang   

  • Received:2008-03-06 Revised:2008-03-28 Online:2009-01-15 Published:2009-01-15

摘要:

目的 探讨人包皮组织来源成黑素细胞的培养条件。方法 包皮取自小儿,采用分离酶和胰酶两步消化法获得表皮细胞悬液,用实验室改进的成黑素细胞培养基培养结合差速黏附法分选成黑素细胞。对培养的成黑素细胞生长情况进行对比观察,并用Dopa染色、TRP2抗体染色、S-100抗体染色以及电镜观察等方法对培养的成黑素细胞进行鉴定。结果 培养的人黑素细胞呈火柴棒状,散在排列,双极对称,胞体瘦小,细胞核折光性强,TRP2抗体染色、S-100抗体染色、Dopa染色均阳性,电镜下可见胞内含有Ⅲ ~ Ⅳ期的成熟黑素颗粒。与黑素细胞相比,培养的人成黑素细胞胞浆丰富,体积较大,呈双极或不规则形,克隆样生长,胞内无Ⅲ ~ Ⅳ期的成熟黑素颗粒,只含有Ⅰ期不成熟黑素颗粒,TRP2抗体染色阳性、S-100抗体染色、Dopa染色阴性。结论 改良的成黑素细胞培养基成功培养出包皮组织来源的成黑素细胞,为表皮色素形成机制的研究打下基础。

关键词: [关键词] 黑素母细胞;体外培养;免疫组化

Abstract:

Objective To study the in vitro culture condition for melanoblasts from human foreskin tissue. Methods The skin tissue taken from foreskin of children was treated with 0.5% dispase II to separate epidermis from dermis, then with trypsin to obtain single cell suspension, which was cultured in modified medium for melanoblasts, i.e., MCDB254 medium supplied with several cell growth factors. Finally, melanoblasts were obtained based on the difference of adhesion speed. The morphology and proliferation of cultured melanoblasts were observed under a light microscope. DOPA staining, immunostaining with anti- S-100 and -tyrosinase related protein 2 (TRP2) antibodies, and transmission electron microscopy were performed to identify the cultured melanoblasts. Results The cultured human melanocytes displayed a match-like shape, scattered arrangement, symmetric double poles, slim cell body, highly refractive nuclei; meanwhile, the melanoblasts exhibited plentiful cytoplasm, large volume, bipolar or irregular shape and clonal growth. Additionally, the melanocytes were positive for TRP2, S-100 and Dopa staining, while the melanoblasts were positive only for TRP2. Electron microscopy revealed the presence of mature melanin granules (stageⅢ - Ⅳ) in melanocytes but immature melanin granules (stageⅠ) in melanoblasts. Conclusion Stable pure culture of melanoblasts has been realized with the reformed medium, which may lay a foundation for the investigation into the mechanism of epidermal pigmentation.

Key words: [Key words] Melanoblasts;In vitro;immunohistochemistry