SQUAMOUS EPITHELIAL CELL INVASION IN ORGAN CULTURE

在拟议的研究中，我们将利用一种新开发的器官 研究人鳞状上皮细胞过程的培养模型 原地条件下的入侵。器官培养模式由2- MM穿孔活检培养始于健康的新生儿包皮(AS 以及来自成人外科标本皮肤)。当纸巾碎片被 在无血清、无生长因子的基础培养基中孵化后，它们仍保持 组织学正常，生化活性长达30天。 然而，当基础培养基加有以下物质的组合时 外源性生长因子包括表皮生长因子、胰岛素和 垂体提取物，基底层角质形成细胞变得多形性和扁平化。 沿着基底膜向外扩散。上皮细胞经历了一个 过度增殖反应和上皮细胞链向下迁移 进入了间充质所占据的空间。尽管最初的衬线是 基底膜，基底膜最终破裂并 上皮细胞穿透间充质。在拟议的研究中，这一点 器官培养模型将用于评估丝氨酸蛋白酶的作用 和基质金属蛋白酶在侵袭过程中的作用。在具体的目标上， 研究将集中在丝氨酸蛋白酶上。在目标的第一部分， 将进行研究，以评估下列情况下的丝氨酸蛋白酶水平 维持正常组织结构的条件 入侵发生的条件。在此之后，一些 介入方法(广谱丝氨酸蛋白酶抑制剂， 纤溶酶和纤溶酶原激活物抑制物、单抗等) 将被用来阻断器官中丝氨酸蛋白酶的表达 文化。这种干预对入侵的影响将是 下定决心。在特定目标II中，重点将放在金属蛋白酶上。 与丝氨酸蛋白酶一样，分析性和干预性研究 将会被执行。通过这种方式，我们希望确定在 丝氨酸蛋白酶和金属蛋白水解酶在口腔鳞癌中的表达 与诱导入侵相结合。更重要的是，通过选择性地 蛋白水解酶的抑制，我们希望能为 特定的酶或酶的级联反应参与入侵 进程。通过研究伴随和贡献的分子事件 在这个模型中，我们希望阐明人类入侵的过程。 鳞状上皮细胞在以下条件下发生侵袭 密切模仿临床环境。

In the proposed studies we will utilize a novel, newly-developed, organ culture model to study the process of human squamous epithelial cell invasion under in situ conditions. The organ culture model consists of 2- mm punch biopsy cultures started from healthy neonatal human foreskin (as well as from adult surgical specimen skin). When the tissue pieces are incubated in a serum-free, growth factor-free basal medium, they remain histologically-normal and biochemically active for up to 30 days. However, when the basal medium is supplemented with a combination of exogenous growth factors including epidermal growth factor, insulin and pituitary extract, the basal keratinocytes become pleomorphic and flatten out along the basement membrane. The epithelial cells undergo a hyperproliferative response and strands of epithelial cells migrate down into the space occupied by the mesenchyme. Although initially lined by basement membrane, the basement membrane eventually ruptures and epithelial cells penetrate the mesenchyme. In the proposed studies, this organ culture model will be used to assess the role of serine proteinases and matrix metalloproteinases in the invasion process. In Specific Aim I, studies will focus on serine proteinases. In the first part of the aim, studies will be carried out to assess levels of serine proteinases under conditions in which normal tissue architecture is maintained and under conditions in which invasion occurs. Following this, a number of interventional approaches (broad spectrum serine proteinase inhibitors, plasmin and plasminogen activator inhibitors, monoclonal antibodies, etc.) will be used to block expression of serine proteinases in the organ cultures. The effects of such interventions on invasion will be determined. In Specific Aim II, the focus will be on metalloproteinases. As with serine proteinases, both analytical and interventional studies will be carried out. In this manner, we hope to identify the changes in serine proteinase and metalloproteinase expression that occur in conjunction with induction of invasion. More importantly, by selective proteinase inhibition, we hope to provide definitive evidence for the involvement of specific enzymes or enzyme cascades in the invasion process. By studying the molecular events that accompany and contribute to invasion in this model, we hope to elucidate the process by which human squamous epithelial cell invasion occurs in man under conditions which mimic closely the clinical setting.