Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue.
Convenient methodology for extraction and subsequent selective propagation of mouse melanocytes in culture from adult mouse skin tissue.
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从成年小鼠皮肤组织中提取培养物并随后选择性繁殖小鼠黑素细胞的便捷方法。
DOI：
10.1016/j.bbrep.2019.100619
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Sakaguchi M
中科院分区：
文献类型：
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作者：
Tomonobu N;Kinoshita R;Sumardika IW;Chen Y;Inoue Y;Yamauchi A;Yamamoto KI;Murata H;Sakaguchi M
小鼠黑色素瘤B16-BL6细胞是研究肿瘤转移的有用细胞。为了在分子水平上理解转移原理，有必要进行比较癌细胞和正常细胞的实验。然而，与正常的人类黑素细胞不同，正常的小鼠黑素细胞的制备是相当困难的，因为缺乏市场和关于建立小鼠黑素细胞原代培养的既定方案的信息不足。本研究旨在建立一种简便的小鼠黑素细胞原代培养方法，并以人黑素细胞原代培养方法为基础。制备纯小鼠黑素细胞的主要障碍是如何消化小鼠皮肤组织，以及如何减少角质形成细胞和成纤维细胞的污染。通过对皮肤标本进行胶原酶消化，短期胰酶消化分离黑素细胞和角质形成细胞，以及在培养液中使用12-O-十四烷基佛波醇13-醋酸酯(TPA)和霍乱毒素，克服了这些障碍。这些补充剂分别起到防止角质形成细胞和成纤维细胞增殖的作用。这一简便的程序使我们能够制备正常小鼠黑素细胞的纯培养。利用富含黑素细胞的正常小鼠黑素细胞和B16-BL6肿瘤细胞，比较黑色素瘤细胞黏附分子(MCAM)在黑色素瘤转移过程中的表达水平。结果表明，MCAM在B16-BL6细胞中的表达明显高于正常小鼠黑素细胞。
Mouse melanoma B16-BL6 cells are useful cells for cancer metastatic studies. To understand the metastatic principle at molecular levels, it is necessary to carry out experiments in which cancer cells and their normal counterparts are compared. However, unlike normal human melanocytes, preparation of normal mouse melanocytes is quite difficult due to the lack of marketing and insufficient information on an established protocol for primary culture of mouse melanocytes. In this study, we aimed to establish a convenient method for primary culture of mouse melanocytes on the basis of the protocol for human melanocytes. The main obstacles to preparing pure mouse melanocytes are how to digest mouse skin tissue and how to reduce the contamination of keratinocytes and fibroblasts. The obstacles were overcome by collagenase digestion for skin specimens, short time trypsinization for separating melanocytes and keratinocytes, and use of 12-O-Tetradecanoylphorbol 13-acetate (TPA) and cholera toxin in the culture medium. These supplements act to prevent the proliferation of keratinocytes and fibroblasts, respectively. The convenient procedure enabled us to prepare a pure culture of normal mouse melanocytes. Using enriched normal mouse melanocytes and cancerous B16-BL6 cells, we compared the expression levels of melanoma cell adhesion molecule (MCAM), an important membrane protein for melanoma metastasis, in the cells. The results showed markedly higher expression of MCAM in B16-BL6 cells than in normal mouse melanocytes.