Abstract：
BACKGROUND: Existing animal models for testing therapeutics in the skin are limited. Mouse and rat models lack similarity to human skin in structure and wound healing mechanism. Pigs are regarded as the best model with regards to similarity to human skin; however, these studies are expensive, time-consuming, and only small numbers of biologic replicates can be obtained. In addition, local-regional effects of treating wounds that are closely adjacent to one-another with different treatments make assessment of treatment effectiveness difficult in pig models. Therefore, here, a novel nude mouse model of xenografted porcine hypertrophic scar (HTS) cells was developed. This model system was developed to test if supplying hypo-pigmented cells with exogenous alpha melanocyte stimulating hormone (α-MSH) will reverse pigment loss in vivo.
METHODS: Dyschromic HTSs were created in red Duroc pigs. Epidermal scar cells (keratinocytes and melanocytes) were derived from regions of hyper-, hypo-, or normally pigmented scar or skin and were cryopreserved. Dermal fibroblasts (DFs) were isolated separately. Excisional wounds were created on nude mice and a grafting dome was placed. DFs were seeded on day 0 and formed a dermis. On day 3, epidermal cells were seeded onto the dermis. The grafting dome was removed on day 7 and hypo-pigmented xenografts were treated with synthetic α-MSH delivered with microneedling. On day 10, the xenografts were excised and saved. Sections were stained using hematoxylin and eosin hematoxylin and eosin (H&E) to assess xenograft structure. RNA was isolated and quantitative real-time polymerase chain reaction (qRT-PCR) was performed for melanogenesis-related genes TYR, TYRP1, and DCT.
RESULTS: The seeding of HTSDFs formed a dermis that is similar in structure and cellularity to HTS dermis from the porcine model. When hyper-, hypo-, and normally-pigmented epidermal cells were seeded, a fully stratified epithelium was formed by day 14. H&E staining and measurement of the epidermis showed the average thickness to be 0.11 ± 0.07 µm vs. 0.06 ± 0.03 µm in normal pig skin. Hypo-pigmented xenografts that were treated with synthetic α-MSH showed increases in pigmentation and had increased gene expression of TYR, TYRP1, and DCT compared to untreated controls (TYR: 2.7 ± 1.1 vs. 0.3 ± 1.1; TYRP1: 2.6 ± 0.6 vs. 0.3 ± 0.7; DCT 0.7 ± 0.9 vs. 0.3 ± 1-fold change from control; n = 3).
CONCLUSIONS: The developed nude mouse skin xenograft model can be used to study treatments for the skin. The cells that can be xenografted can be derived from patient samples or from pig samples and form a robust dual-skin layer containing epidermis and dermis that is responsive to treatment. Specifically, we found that hypo-pigmented regions of scar can be stimulated to make melanin by synthetic α-MSH in vivo.
METHODS: Dyschromic HTSs were created in red Duroc pigs. Epidermal scar cells (keratinocytes and melanocytes) were derived from regions of hyper-, hypo-, or normally pigmented scar or skin and were cryopreserved. Dermal fibroblasts (DFs) were isolated separately. Excisional wounds were created on nude mice and a grafting dome was placed. DFs were seeded on day 0 and formed a dermis. On day 3, epidermal cells were seeded onto the dermis. The grafting dome was removed on day 7 and hypo-pigmented xenografts were treated with synthetic α-MSH delivered with microneedling. On day 10, the xenografts were excised and saved. Sections were stained using hematoxylin and eosin hematoxylin and eosin (H&E) to assess xenograft structure. RNA was isolated and quantitative real-time polymerase chain reaction (qRT-PCR) was performed for melanogenesis-related genes TYR, TYRP1, and DCT.
RESULTS: The seeding of HTSDFs formed a dermis that is similar in structure and cellularity to HTS dermis from the porcine model. When hyper-, hypo-, and normally-pigmented epidermal cells were seeded, a fully stratified epithelium was formed by day 14. H&E staining and measurement of the epidermis showed the average thickness to be 0.11 ± 0.07 µm vs. 0.06 ± 0.03 µm in normal pig skin. Hypo-pigmented xenografts that were treated with synthetic α-MSH showed increases in pigmentation and had increased gene expression of TYR, TYRP1, and DCT compared to untreated controls (TYR: 2.7 ± 1.1 vs. 0.3 ± 1.1; TYRP1: 2.6 ± 0.6 vs. 0.3 ± 0.7; DCT 0.7 ± 0.9 vs. 0.3 ± 1-fold change from control; n = 3).
CONCLUSIONS: The developed nude mouse skin xenograft model can be used to study treatments for the skin. The cells that can be xenografted can be derived from patient samples or from pig samples and form a robust dual-skin layer containing epidermis and dermis that is responsive to treatment. Specifically, we found that hypo-pigmented regions of scar can be stimulated to make melanin by synthetic α-MSH in vivo.
摘要：
背景:用于测试皮肤中的治疗剂的现有动物模型是有限的。小鼠和大鼠模型在结构和伤口愈合机制上与人类皮肤缺乏相似性。就与人类皮肤的相似性而言，猪被认为是最好的模型；然而，这些研究是昂贵的，耗时,只能获得少量的生物重复。此外,用不同的治疗方法治疗彼此紧密相邻的伤口的局部区域效应使得在猪模型中难以评估治疗效果。因此,在这里，建立了一种新型的猪增生性瘢痕（HTS）细胞裸鼠模型。开发此模型系统是为了测试向色素沉着不足的细胞提供外源性α黑素细胞刺激激素（α-MSH）是否会逆转体内色素损失。
方法:在红色Duroc猪中产生变色异常HTSs。表皮瘢痕细胞（角质形成细胞和黑素细胞）来自高，假设-，或通常有色素的疤痕或皮肤，并冷冻保存。分别分离真皮成纤维细胞(DF)。在裸小鼠上产生切除伤口并放置移植圆顶。在第0天接种DF并形成真皮。在第3天,将表皮细胞接种到真皮上。在第7天移除移植穹顶,并用微针递送的合成α-MSH处理色素沉着不足的异种移植物。在第10天,切除并保存异种移植物。使用苏木精和伊红苏木精和伊红(H&E)对切片进行染色以评估异种移植物结构。分离RNA，并对黑素生成相关基因TYR进行定量实时聚合酶链反应（qRT-PCR）。TYRP1和DCT。
结果:HTSDF的接种形成了与来自猪模型的HTS真皮在结构和细胞性方面相似的真皮。当hyper-，假设-，正常色素的表皮细胞被接种，在第14天形成完全复层的上皮.H&E染色和表皮测量显示平均厚度为0.11±0.07µm正常猪皮中的0.06±0.03µm。用合成α-MSH处理的色素沉着不足的异种移植物显示色素沉着增加并且TYR的基因表达增加。与未治疗对照相比，TYRP1和DCT(TYR：2.7±1.1vs.0.3±1.1;TYRP1:2.6±0.6vs.0.3±0.7;DCT0.7±0.9vs.与对照组相比变化0.3±1倍；n=3）。
结论:开发的裸鼠皮肤异种移植模型可用于研究对皮肤的治疗。可以异种移植的细胞可以来源于患者样品或猪样品,并形成对治疗有响应的含有表皮和真皮的坚固的双皮肤层。具体来说,我们发现，在体内合成α-MSH可以刺激瘢痕色素沉着不足的区域产生黑色素。
方法:在红色Duroc猪中产生变色异常HTSs。表皮瘢痕细胞（角质形成细胞和黑素细胞）来自高，假设-，或通常有色素的疤痕或皮肤，并冷冻保存。分别分离真皮成纤维细胞(DF)。在裸小鼠上产生切除伤口并放置移植圆顶。在第0天接种DF并形成真皮。在第3天,将表皮细胞接种到真皮上。在第7天移除移植穹顶,并用微针递送的合成α-MSH处理色素沉着不足的异种移植物。在第10天,切除并保存异种移植物。使用苏木精和伊红苏木精和伊红(H&E)对切片进行染色以评估异种移植物结构。分离RNA，并对黑素生成相关基因TYR进行定量实时聚合酶链反应（qRT-PCR）。TYRP1和DCT。
结果:HTSDF的接种形成了与来自猪模型的HTS真皮在结构和细胞性方面相似的真皮。当hyper-，假设-，正常色素的表皮细胞被接种，在第14天形成完全复层的上皮.H&E染色和表皮测量显示平均厚度为0.11±0.07µm正常猪皮中的0.06±0.03µm。用合成α-MSH处理的色素沉着不足的异种移植物显示色素沉着增加并且TYR的基因表达增加。与未治疗对照相比，TYRP1和DCT(TYR：2.7±1.1vs.0.3±1.1;TYRP1:2.6±0.6vs.0.3±0.7;DCT0.7±0.9vs.与对照组相比变化0.3±1倍；n=3）。
结论:开发的裸鼠皮肤异种移植模型可用于研究对皮肤的治疗。可以异种移植的细胞可以来源于患者样品或猪样品,并形成对治疗有响应的含有表皮和真皮的坚固的双皮肤层。具体来说,我们发现，在体内合成α-MSH可以刺激瘢痕色素沉着不足的区域产生黑色素。
