Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers.
Skin Immuno-CometChip in 3D vs. 2D Cultures to Screen Topical Toxins and Skin-Specific Cytochrome Inducers.
复制标题
皮肤免疫彗星芯片在 3D 与 2D 培养中筛选局部毒素和皮肤特异性细胞色素诱导剂。
作者：
局部基因毒剂的目标是皮肤的基底细胞和干细胞。这些细胞可能会错误修复DNA损伤，导致肿瘤抑制基因或癌基因的有害突变。然而，许多化合物的遗传毒性尚未确定，需要使用相关的皮肤模型进行测试。为此，我们设计了一种新的高通量检测方法，用于检测在表皮干细胞和基底细胞中造成DNA损伤的试剂，并用它来测试已知的DNA损伤剂。我们利用2D表皮细胞或3D皮肤等效物，将它们局部暴露于不同的化合物中。皮肤免疫芯片检测使用在胶原/琼脂糖混合物中形成的微孔阵列，通过胶原与仅存在于基底细胞和干细胞上的α2β1整合素结合来捕获每个微孔中的单个基底细胞。免疫荧光标记细胞证实了β-1整合素的存在，然后将细胞置于电场中，允许NiCDNA在碱性条件下从类核细胞中迁移出来，并对所产生的DNA彗星进行染色和成像。此外，使用改进的彗星探测软件可以自动和快速地量化DNA损伤。我们的研究表明，通过使用3D皮肤培养以及2D单层培养的角质形成细胞，我们可以准确地预测遗传毒性。
The targets of topical genotoxic agents are basal and stem cells of the skin. These cells may misrepair DNA lesions, resulting in deleterious mutations of tumor suppressors or oncogenes. However, the genotoxicity of many compounds has not as yet been determined and needs to be tested using a relevant skin model. To this end, we designed a new high-throughput assay for the detection of agents that create DNA damage in epidermal stem and basal cells and used it to test known DNA-damaging agents. We utilized either 2D epidermal cells or 3D skin equivalents and topically exposed them to different compounds. The Skin Immuno-CometChip assay uses arrays of microwells formed in a collagen/agarose mixture to capture single basal cells in each microwell by virtue of collagen binding to α2β1 integrin, which is present only on basal and stem cells. The presence of β1 integrin was verified by immunofluorescent labeling cells that were then subjected to an electrical field, allowing for the migration of nicked DNA out of the nucleoid in alkali, with the resulting DNA comets stained and imaged. Furthermore, using improved comet detection software allowed for the automated and rapid quantification of DNA damage. Our study indicates that we can accurately predict genotoxicity by using 3D skin cultures, as well as keratinocytes grown in 2D monolayers.
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影响因子：
3.7
作者：
Acheva A;Ghita M;Patel G;Prise KM;Schettino G
通讯作者：
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影响因子：
19
作者：
Huh, Dongeun;Hamilton, Geraldine A.;Ingber, Donald E.
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DOI：
10.1016/s0027-5107(97)00306-0
发表时间：
1998-06-18
影响因子：
2.3
作者：
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120.7
作者：
Matta, Murali K.;Florian, Jeffry;Strauss, David G.
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影响因子：
6.5
作者：
Baron, JM;Höller, D;Jugert, FK
通讯作者：
Jugert, FK