CULTURED EPIDERMAL GRAFTS FOR BURN PATIENTS

这项提议的最终目标是开发出能够 用于生产培养的人表皮细胞的移植物 它将提供最佳的覆盖率和快速的烧伤愈合 伤口。为了制定程序来扩展我们的能力 为了提供这种培养的表皮，我们已经在 更详细地说，生长和分化的动力学 按培养方法培养的人角质形成细胞 它是我们开发的(1，2)。这些研究的结果增加了 我们建立培养移植物的能力，现在可以使用 常规促进烧伤创面快速愈合(1，3，4)。我们 建议开发增加养殖供给的手段 表皮细胞通过细胞片的低温“堆积”进行移植。 我们还将测试这样一种假设，即细胞培养已经 移植前体外诱导形成角质层 可提供比烧伤创面更快的覆盖和愈合 根据我们的标准培养的分化程度较低的细胞片 培养方法。具体目标是： I.量化冷冻保存对以下各项的影响： 角质形成细胞悬液的生长和成熟动力学。 B.培养的细胞片发挥作用的能力 体内移植。 二、发展促进植物终末分化的方法 培养中的表皮细胞。我们的努力将集中在 利用下列化学和/或无毒生物制剂 可作为中等补充剂使用。初步实验 提示N-丁酸钠(NAB)可增强末端 角质形成细胞在短期培养中的分化 一段时间。这些研究将会扩展至： A.描述细胞亚群和生长动力学 角质形成细胞诱导形成去核的角质形成细胞。 B.量化细胞骨架蛋白的表达和细胞 体外分化至去核过程中的组织 角膜细胞。 C.检查培养的细胞片的能力，其中包含一个 角质层细胞层起到体内移植的作用。

The ultimate goal of this proposal is to develop methods that can be applied to produce grafts of cultured human epidermal cells which will provide optimal coverage and rapid healing of burn wounds. In order to develop procedures for expanding our ability to provide such cultured epidermis, we have characterized, in detail, the kinetics of growth and differentiation which occur in cultures of human keratinocytes grown according to a culture method which we developed (1,2). The results of these studies increased our ability to establish cultured grafts which can now be used routinely to promote the rapid healing of burn wounds (1,3,4). We propose to develop the means of increasing the supply of cultured epidermal cell grafts through cryogenic "banking" of cell sheets. We will also test the hypothesis that cell cultures which have been induced to form a stratum corneum in vitro prior to transplantation can provide more rapid coverage and healing of burn wounds than less differentiated cell sheets grown according to our standard cultures methods. The specific aims are: I. To quantitate the effects of cryopreservation on: A. The growth and maturation kinetics of keratinocyte suspensions. B. The ability of the cultured cell sheets to function as transplants in vivo. II. To develop methods to promote the terminal differentiation of epidermal cells in culture. Our efforts will focus on the utilization of chemical and/or non-toxic biological agents which can be used as medium supplements. Preliminary experiments indicate that sodium-N-butyrate (NaB) can enhance terminal differentiation of keratinocytes when added to culture for a short period of time. These studies will be expanded to: A. Characterize the cell subpopulation and growth kinetics of keratinocytes induced to form enucleated corneocytes. B. Quantitate cytoskeletal protein expression and cellular organization during in vitro differentiation to enucleated corneocytes. C. Examine the ability of cultured cell sheets which contain a stratum corneum cell layer to function as transplants in vivo.