Use of organotypic coculture to study keloid biology
Use of organotypic coculture to study keloid biology
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DOI：
10.1016/j.amjsurg.2007.10.003
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发表时间：
2008-02-01
影响因子：
3
通讯作者：
Yang, George P.
中科院分区：
文献类型：
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作者：
Butler, Paris D.;Ly, Daphne P.;Yang, George P.
背景：瘢痕疙瘩是一种病理性疤痕，困扰着很大一部分人，目前尚无明确的治疗方法。瘢痕疙瘩形成的动物模型的缺乏阻碍了研究。我们建立了一个体外器官型皮肤模型来模拟正常瘢痕疙瘩生物学，这可能使我们在没有动物模型的情况下研究瘢痕疙瘩的形成。方法：在胶原基质中培养正常（NFs）和瘢痕疙瘩（KFs）人成纤维细胞，形成三维真皮结构。正常的人角质形成细胞（NKs）被培养为顶部的第二层，并暴露在空气-流体界面中，使其分化为成熟的角质形成细胞层。器官型皮肤在含有10%胎牛血清的Dulbecco改良鹰培养基中保存28天。样品采集，处理，切片，苏木精和伊红染色，然后进行定性分析。免疫印迹法测定a-平滑肌肌动蛋白。结果：与NF/NK型皮肤相比，KF/NK型皮肤显示胶原沉积增加，胶原染色明显致密，真皮厚度增加。我们看到KF/NK结构的挛缩增加，与NF/NK皮肤相比，这与KF/NK器官型皮肤真皮层中a-平滑肌肌动蛋白纤维的组织增加有关。结论：我们已经证明，与NFs和NKs相比，KFs与瘢痕疙瘩角质形成细胞共培养导致胶原生成增加和皮肤挛缩，这与已知的瘢痕疙瘩行为一致。由于缺乏动物模型，我们认为器官型皮肤培养可以作为研究瘢痕疙瘩形成的替代方法。(C) 2008节选医药股份有限公司版权所有。
Background: Keloids are pathologic scars afflicting a large segment of our population and for which there is no definitive therapy. The lack of an animal model for keloid formation has hampered study. We developed an in vitro organotypic skin model to simulate normal keloid biology, which may allow us to study keloid formation without an animal model.Methods: Normal (NFs) and keloid (KFs) human fibroblasts were cultured in a collagen matrix to create a 3-dimensional dermal structure. Normal human keratinocytes (NKs) were cultured as a second layer on top and exposed to an air-fluid interface to allow differentiation into a mature keratinocyte layer. The organotypic skin was maintained for 28 days in Dulbecco's modified eagle medium with 10% fetal calf serum. Samples were collected, processed, sectioned, stained with hematoxylin and eosin, and then measured for qualitative analysis. a-smooth-muscle actin was also evaluated by immunoblotting.Results: KF/NK organotypic skin showed increased collagen deposition, based on significantly denser collagen staining, with increased dermal thickness compared with NF/NK organotypic skin. We saw increased contracture in the KF/NK construct, and this correlated with increased organization of a-smooth-muscle actin fibers in the dermal layer of KF/NK organotypic skin compared with NF/NK skin.Conclusions: We have shown that coculture of KFs with keloid keratinocytes leads to an increased collagen production and dermal contracture compared with NFs and NKs, consistent with known keloid behavior. Given the lack of an animal model, we believe that organotypic skin culture can serve as a surrogate to study keloid formation. (C) 2008 Excerpta Medica Inc. All rights reserved.