建立临床相关<em>离体</em>调查上皮创伤修复的微环境本地人模拟白内障手术模型

Summary

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

Abstract

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells’ native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens¹. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Introduction

临床相关，模拟白内障手术，这里所描述的体外上皮伤口愈合模型的开发是为了提供调查，在响应于损伤调节修复上皮组织的机制的一种工具。在创建该模型旨在为关键特征包括1）提供条件精密地复制在体内响应于伤人在培养设置，2）缓和调制修复的调控元件，以及3）能力图像的修复过程中，其整体，在实际时间。我们面临的挑战，因此，是在细胞的天然微环境创造一种文化模式，即有可能学习和操作，上皮创面修复。这种创伤修复模型的有效性开辟了新的可能性，标识来自调节修复过程基质蛋白，细胞因子和趋化因子的内源性信号提示。另外，该模型是理想的研究如何一Ñ ​​上皮能够移动作为集体片重新epithelialize伤口区域^2,3，和用于确定在伤口边缘起作用的引导受伤上皮⁴的集体迁移间充质领袖细胞的谱系。这种模式还提供了一个用以标识疗法，可以有效的促进伤口愈合，防止异常创面修复⁵的平台。

目前已经有许多可用的创伤修复模式，无论是在文化和体内 ，它提供了当今大多数所谓对伤口修复过程。在动物损伤模型，如角膜^6-12和皮肤^13-17，有研究的组织的响应伤人的所有修复介质是可以参与的过程中，包括来自所述的上下文中，机会血管和神经系统。但是，也有限制操纵experi体内的精神的条件下，它是目前无法进行体内修复反应的成像研究，随时间连续。与此相反，大多数体外伤口修复培养模型，如划痕，可以很容易地操纵和随后随着时间的推移，但缺乏研究伤口愈合中的体内组织的环境背景。虽然体外模型提供了细胞的微环境加上调节修复的分子调节的过程中的任何时间点的能力范围内不断研究损伤修复过程中随着时间的优势，很少有车型适合这些参数。

这里描述的方法，以产生高度可重复的离体上皮的伤口愈合是再现上皮组织的响应于生理伤人培养物。使用鸡胚镜头作为组织源， 体外 MOCķ白内障手术被执行。该透镜是一种理想的组织用于这些研究，因为它是自包含在一个厚的基底膜胶囊，无血管，没有神经支配，且没有任何相关联的基质^18,19。在人类疾病，白内障手术涉及视力丧失，由于透镜的混浊，并且包括除去晶状体纤维细胞块，其包含大量的透镜组成。白内障手术后视力是通过人工眼内透镜的插入恢复。在白内障手术过程中，通过除去纤维细胞，诱导在相邻透镜上皮，这是为了响应通过再上皮晶状体囊的后部区域已被占用的纤维细胞的损伤的反应。在白内障手术中，因为在大多数伤口修复反应，还有，有时会发生的异常纤维化结果向伤口愈合反应，与肌纤维母细胞的出现，这在透镜被称为后验Capsu相关联乐混浊^20-22。为了产生白内障手术伤口愈合的模型，一个白内障手术的过程是模仿从鸡胚眼中除去产生生理损伤镜头。显微手术切除晶状体纤维细胞导致的晶状体上皮细胞包围着一个非常一致的圆形伤口面积。该细胞群保持牢固地附着在透镜基底膜胶囊和由外科手术过程中受伤。上皮细胞迁移到内源性基底膜的裸露面积愈合伤口的带动下，在维修过程中被称为领导者细胞¹波形丰富的间充质细胞群。用该模型的上皮损伤的响应可容易地可视化并随后随时间在细胞的微环境的上下文中。细胞是易于接触到的表达或活化有望发挥在伤口修复中的作用的分子的修饰。日的一项强大功能是模型是分离和研究在伤口愈合的框架迁移特异性改变的能力。准备大量老年匹配体外伤口愈合培养物用于研究的能力是该模型的另一优点。因此，这个模型系统提供了一个独特的机会，梳理出创面修复机制和试验治疗为他们的伤口愈合过程中的作用。预计在体外模拟白内障手术模式，具有广泛的适用性，为研究损伤修复机制的重要资源。

Protocol

下面的协议符合托马斯·杰斐逊大学机构动物护理和使用委员会的指导方针，并与ARVO声明为动物的视觉研究使用。 1.安装镜头，并准备于离体培养伤口将3座100 25mm培养皿在无菌的层流罩。填充两个的中途用Tris /葡萄糖缓冲培养皿（TD缓冲器; 140 mM氯化钠，5mM的氯化钾，0.7毫的Na 2 PO 4，5mM的D-葡萄糖，8.25毫摩尔Tris碱，pH值至7.4，用HCl）在RT ，留下?…

Representative Results

离体模型创建研究伤口愈合过程中的细胞的天然微环境调查涉及细胞的微环境原生内调节上皮伤口愈合机制，创建了一个临床相关的体外模拟白内障手术模式。这个模型是从晶状体组织提供了许多优点，由于其内在特性创建：1）透镜是一个自包含的器官由粗基底膜称为晶状体囊包围; 2）它是无血管，3）没有神经支配和4）免费相关基质。因此，检查了修复过程?…

Discussion

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues^2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni…

Materials

Sodium Chloride (NaCl)	Fisher Scientific	S271-3	Use at 140mM in TD Buffer
Potassium Chloride (KCl)	Fisher Scientific	P217-500	Use at 5mM in TD Buffer
Sodium Phosphate (Na2HPO4)	Sigma	S0876	Use at .7mM in TD Buffer
D-glucose (Dextrose)	Fisher Scientific	D16-500	Use at 0.5mM in TD Buffer
Tris Base	Fisher Scientific	BP152-1	Use at 8.25mM in TD Buffer
Hydrochloric acid	Fisher Scientific	A144-500	Use to pH TD buffer to 7.4
Media 199	GIBCO	11150-059
L-glutamine	Corning/CellGro	25-005-CI	Use at 1% in Media199
Penicillin/streptomycin	Corning/CellGro	30-002-CI	Use at 1% in Media199
100mm petri dishes	Fisher Scientific	FB0875711Z
Stericup Filter Unit	Millipore	SCGPU01RE	Use to filter sterilize Media
Dumont #5 forceps (need 2)	Fine Science Tools	11251-20
35mm Cell Culture Dish	Corning	430165
27 Gauge 1mL SlipTip with precision glide needle	BD	309623
Fine Scissors	Fine Science Tools	14058-11
Standard Forceps	Fine Science Tools	91100-12
Other Items Needed: General dissection instruments,  fertile white leghorn chicken eggs,
check egg incubator (humidified, 37.7°C), laminar flow hood, binocular stereovision dissecting
microscope