Polyvalent protein-ligand displays for human endometrial stromal cell and embryonic stem cell adhesion differentiation and proliferation

有时需要器官移植来替代疾病或功能失调的组织，但患者必须服用免疫抑制剂，这有令人不快的副作用，以防止移植组织的排斥反应。这个项目的目的是通过一个叫做组织工程的过程来发现解决这个问题的方法。组织工程的目标之一是从患者的健康组织中提取细胞，并在实验室（体外）扩增该组织，以便移植回患者体内，而不必担心免疫反应。在体内，细胞粘附在由蛋白质组成的细胞外基质上，这些蛋白质指导细胞发育（分化）和组织，这是组织工程中成功扩展组织的先决条件。许多细胞与一种叫做纤维连接蛋白的细胞外基质蛋白相互作用。由于在实验室中需要合成基质来支持细胞，我们需要能够模拟纤维连接蛋白。这通常是通过一种叫做RGD的小分子来完成的，这种小分子来源于纤维连接蛋白。然而，RGD不能提供通常由纤维连接蛋白提供的全部细胞支持。为了克服这一限制，我们打算使用与细胞充分相互作用的纤维连接蛋白片段。这些纤维连接蛋白片段将以特定的方向（从侧面看）和模式（从上面看）附着在表面上。预期的模式将显示纤维连接蛋白碎片均匀分布在整个表面。我们将从子宫内膜组织中制备人类细胞，因为这些细胞在实验室中生长良好且可复制。我们将利用我们改良的表面对细胞进行培养，从而研究在人工细胞支架中控制细胞粘附和分化的机制。然后，我们打算研究三维细胞生长（通常在组织组织过程中发生），通过将纤维连接蛋白片段附着在凝胶状物质中制作3D支架。人体细胞在3D支架内的成功增殖和分化将是许多组织类型工程用于医学的第一步。组织工程的一个重要策略是使用胚胎干细胞，这是不寻常的，因为它们有可能特化成任何特定的细胞类型（如心肌、皮肤或子宫内膜）。当不能从病人身上取出组织进行体外扩增时，干细胞是有价值的。它们还能让我们产生原本只能从动物身上提取的组织。我们的工作将研究最初来源于小鼠胚胎的干细胞如何在纤维连接蛋白片段修饰的表面上繁殖和分化。这些小鼠干细胞在明胶支架上生长（通常情况下是这样），分化成血细胞、心脏细胞或血管内膜细胞。因此，我们的工作将在显微镜下专门寻找这些类型的成熟细胞和与不同细胞类型相关的基因。通过这种方式，可以评估纤维连接蛋白对干细胞的影响，这将是确定可能用于组织生成的培养条件的重要一步。

Organ transplants are sometimes required to replace diseases or dysfunctional tissue but patients must take immune suppressants, which have unpleasant side-effects, to prevent rejection of the transplanted tissue. The aim of this project is to discover ways of getting around this problem by a process called tissue engineering. One of the goals of tissue engineering is to take cells from the patient¿s healthy tissue and expand that tissue in the laboratory (in vitro) for transplantation back into the patient without fear of immune response. In the body cells stick (adhere) to an extracellular matrix composed of proteins which guide the cells development (differentiation) and organisation, which are prerequisites for successful expansion of tissue in tissue engineering. Many cells interact with an extracellular matrix protein called fibronectin. Since a synthetic matrix is required for cell support in the laboratory we need to be able to mimic fibronectin. This is commonly done using a small molecular called RGD derived from fibronectin. However, RGD does not provide the full cell support normally afforded by fibronectin. To overcome this limitation we intend to use fragments of fibronectin which fully interact with cells. These fibronectin fragments will be attached to surfaces in a specific orientation (as viewed from the side) and pattern (as viewed from above). The intended pattern will show clusters of fibronectin fragments evenly distributed across the surface. We will prepare human cells from the tissue lining the womb since these cells grow well and reproducibly in the laboratory. We will incubate the cells with our modified surfaces so that we can study the mechanisms controlling cell adherence and differentiation in the artificial cell supports. We then intend to investigate cell growth in three-dimensions (as would normally occur during the organisation of tissue), making 3D scaffolds by attaching the fibronectin fragments within a gel-like substance. Successful proliferation and differentiation of human cells within the 3D scaffold would be a first step to engineering of many tissue type for use in medicine. An important strategy for tissue engineering is the use of embryonic stem cells, which are unusual in that they have the potential to specialise into any particular cell type (such as heart muscle, skin or endometrium). Stem cells are of value where tissue cannot be taken from the patient for in vitro expansion. They would also allow us to generate tissue that would otherwise have to come from animals for experiments. Our work will study how stem cells, originally derived from mouse embryos, multiply and differentiate on surfaces modified with the fibronectin fragments. These mouse stem cells when grown on gelatine supports (as is normally the case) differentiate into either blood cells, heart cells or cells lining the blood vessels. Our work will therefore look specifically for these types of mature cells under the microscope and genes associated with the different cell types. In this way the impact of fibronectin on the stem cells can be assessed and will represent an important step in defining culture conditions that may be of use in tissue generation.