Isolation, culture and phenotypic characterization of human sweat gland epithelial cells.
Isolation, culture and phenotypic characterization of human sweat gland epithelial cells.
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DOI：
10.3892/ijmm.2014.1851
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发表时间：
2014-10
影响因子：
5.4
通讯作者：
Li Y
中科院分区：
文献类型：
--
作者：
Gao Y;Li M;Zhang X;Bai T;Chi G;Liu JY;Li Y
汗腺上皮细胞（SGEC）已被确定为汗腺再生和含有皮肤附件的皮肤替代品的建设所必需的。因此，SGEC的分离、培养和表型表征至关重要。在本研究中，人的汗腺是通过移液分离后，在相差显微镜消化胶原酶I型。随后，显微器官培养技术用于人SGEC的原代培养，并修改培养条件以达到最佳细胞生长状态。基于其对汗腺特异性标志物的表达来鉴定原代SGEC，所述标志物包括癌胚抗原（CEA）、CK 7、CK 8、CK 14、CK 15、CK 18和CK 19。我们探讨了干细胞在人类汗腺中的可能存在，通过检测其富含亮氨酸重复的G蛋白偶联受体5（LGR 5）的表达。在无血清条件下培养时，原代SGEC达到良好的生长状态。细胞在含1%胎牛血清的无血清培养液中培养1代后仍有明显的增殖活性。通过免疫荧光显微镜、逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析进行的表型分析证实了汗腺特异性标志物的表达，包括CEA、CK 7、CK 8、CK 14、CK 15、CK 18和CK 19。RT-PCR和免疫组化检测LGR 5的表达。与传统的含血清条件相比，无血清培养为人SGEC提供了优选的培养条件。LGR 5是一种识别人类汗腺来源干细胞的新标记物。
Sweat gland epithelial cells (SGECs) have been identified as essential for the regeneration of sweat glands and for the construction of skin substitutes containing skin appendages. Consequently, the isolation, culture and phenotypic characterization of SGECs are of paramount importance. In the present study study, human sweat glands were isolated by pipetting under a phase contrast microscope following digestion with collagenase type I. Subsequently, a microscopic organ culture technique was used for the primary culture of human SGECs, and the culture conditions were modified in order to achieve optimal cell growth status. Primary SGECs were identified based on their expression of markers specific for sweat glands, including carcinoembryonic antigen (CEA), CK7, CK8, CK14, CK15, CK18 and CK19. We explored the possible presence of stem cells in human sweat glands by detecting their expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). Primary SGECs achieved a good growth state when cultured under serum-free conditions. After one passage, the cells cultured in keratinocyte serum-free medium with 1% fetal bovine serum (FBS) still showed a prominent proliferative activity. Phenotypic analysis by immunofluorescence microscopy, reverse transcription-polymerase chain reaction (RT-PCR), and western blot analysis demonstrated the expression of sweat gland-specific markers, including CEA, CK7, CK8, CK14, CK15, CK18 and CK19. In addition, RT-PCR and immunochemistry detected the expression of LGR5. In comparison with traditional serum-containing conditions, serum-free culture provides the preferred culture conditions for human SGECs. LGR5 is a novel marker that identifies human sweat gland-derived stem cells.
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影响因子：
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