Establishment of human skin organotypic cultures (skin-equivalents) as suitable bioassay models and investigation of dermal-epidermal interactions by using the culture system

建立人类皮肤器官培养物（皮肤等效物）作为合适的生物测定模型，并使用培养系统研究真皮-表皮相互作用

基本信息

批准号：
16380227
负责人：
NISHIYAMA Toshio
金额：
$ 9.92万
依托单位：
Tokyo University of Agriculture and Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

In vitro models of human skin utilizing keratinocytes grown upon fibroblast-contracted collagen gels have been known to demonstrate similar behavior to the cells in vivo. The purpose of our study is to provide suitable bioassay models for investigating the structure and function of epidermal or dermal tissue and to determine the dermal-epidermal interactions required for keratinocyte function, basement membrane formation and hair follicle formation utilizing the three-dimensional organotypic cultures such as skin-equivalents (SEs). Despite production of the epidermal basement membrane (BM) components, BM was rarely observed in SEs. Analyses through SEs have revealed that matrix metalloproteinases (MMPs) and plasmin are involved in BM formation. To elucidate the affect of these proteinases, we examined effects of inhibitors for MMPs and serine proteinase on the BM formation and the epidermal structure and differentiation in the SEs. By addition of both inhibitors the epidermis was well … More organized with multilayered keratinocytes consisting of basal, spinous, granular, and cornified layers. The epidermal differentiation markers were expressed in the upper layer of the epidermis and the sharply defined depositions specific for BM components were observed at the dermal-epidermal junction. Ultrastructurally, the extended and continuous lamina densa with approximately 30 nm thick was observed underneath the basal keratinocytes. These results suggest that the assembly of BM under the basal keratinocytes and the differentiation of epidermis are extensively promoted under the influence of inhibitors for MMPs and serine proteinases. In attempt on hair follicle formation by using this culture system, mouse dermal papilla cells were cultured at the dermal-epidermal interface in SEs. Cellular masses including the dermal papilla cells were frequently observed in the upper part of the dermis. Further studies are needed to determine whether the cellular masses are similar to primitive hair follicles. Less

已知利用在成纤维细胞收缩的胶原凝胶上生长的角质细胞的体外模型表现出与体内细胞相似的行为。我们研究的目的是提供合适的生物测定模型来研究表皮或真皮组织的结构和功能。并利用三维器官型培养物（例如皮肤等效物）确定角质形成细胞功能、基底膜形成和毛囊形成所需的真皮-表皮相互作用尽管产生了表皮基底膜 (BM) 成分，但在 SE 中很少观察到 BM。通过 SE 的分析表明，基质金属蛋白酶 (MMP) 和纤溶酶参与了 BM 的形成。我们检查了 MMP 和丝氨酸蛋白酶抑制剂对 BM 形成以及表皮结构和 SE 分化的影响。通过添加两种抑制剂，表皮状况良好。 …更多由基底层、棘层、颗粒层和角化层组成的多层角质形成细胞组成，表皮分化标记物在表皮上层表达，并且在真皮-表皮交界处观察到特定于 BM 成分的清晰沉积。，在基底角质形成细胞下方观察到延伸且连续的厚度约为30 nm的致密层，这些结果表明基底角质形成细胞下方的BM组装。在 MMP 和丝氨酸蛋白酶抑制剂的影响下，角质形成细胞和表皮的分化得到广泛促进。在尝试使用该培养系统形成毛囊时，在 SE 的真皮-表皮界面处培养了小鼠真皮乳头细胞。真皮上部经常观察到真皮乳头细胞，以确定细胞团是否与原始毛囊相似。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG)

使用卵泡抑素相关基因 (FLRG) 的第二个卵泡抑素结构域纯化重组激活素 A

DOI：
发表时间：
2006
期刊：
Protein Expression and Purification 49(1)
影响因子：
0
作者：
Amano S;Ogura Y;Akutsu N;Nishiyama T;Koji Arai
通讯作者：
Koji Arai

Quantitative analysis of laminin 5 gene expression in human keratinocytes

人角质形成细胞中层粘连蛋白5基因表达的定量分析

DOI：
发表时间：
2005
期刊：
Experimental Dermatology 14
影响因子：
0
作者：
Arai KY;Tsuchida K;Li C;Watababe G;Sugino H;Taya K;Nishiyama T;Nobuko Akutsu
通讯作者：
Nobuko Akutsu

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay

采用灵敏夹心酶联免疫法定量分析培养的人真皮成纤维细胞中 VII 型胶原的合成和分泌