SQUAMOUS EPITHELIAL CELL INVASION IN ORGAN CULTURE

在拟议的研究中，我们将利用一种新的，新开发的器官， 培养模型，以研究人鳞状上皮细胞 在原位条件下的侵入。 器官培养模型由2- 从健康的新生儿人包皮开始（作为 以及来自成人外科标本皮肤）。 当组织碎片 在无血清、无生长因子的基础培养基中孵育， 组织学正常且生物化学活性长达30天。 然而，当基础培养基补充有以下组合时， 外源性生长因子，包括表皮生长因子、胰岛素和 垂体提取物，基底角质形成细胞变得多形性和扁平 沿着基底膜。 上皮细胞经历一个 过度增殖反应和上皮细胞链向下迁移 进入间充质所占据的空间。 虽然最初由 基底膜，基底膜最终破裂， 上皮细胞穿透间质。 在这些研究中， 器官培养模型将用于评估丝氨酸蛋白酶的作用 和基质金属蛋白酶的作用。 在具体目标I中， 研究将集中于丝氨酸蛋白酶。 在目标的第一部分中， 将进行研究以评估丝氨酸蛋白酶的水平， 在维持正常组织结构的条件下， 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剂、单克隆抗体等） 将用于阻断器官中丝氨酸蛋白酶的表达 cultures. 这种干预对入侵的影响将是 测定 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，分析和干预研究 将被执行。 通过这种方式，我们希望确定 丝氨酸蛋白酶和金属蛋白酶的表达， 与诱导入侵的结合。 更重要的是，选择性 蛋白酶抑制，我们希望提供明确的证据， 特定酶或酶级联参与入侵 过程 通过研究伴随并促成 在这个模型中，我们希望阐明人类入侵的过程， 鳞状上皮细胞的侵袭发生在人类中， 模拟临床环境。

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.