Mechanisms of cell membrane repair in corneal cells

本研究的长远目标是了解角膜细胞细胞膜修复的基本机制。需要验证的主要假设是，角膜的细胞膜修复依赖于囊泡运输，其中囊泡的招募、运输、胞吐作用在破坏部位附近的对接和融合对于角膜细胞膜的再密封是必不可少的。这一假设将通过监测微穿刺伤口后的膜重封、胞吐和膜张力来检验。胞外融合将被神经毒素破坏已知胞外作用中囊泡融合所需的SNARE复合物的形成而破坏。特异性激酶抑制剂和激活剂将用于阐明钙/钙调素依赖性蛋白激酶（CaM激酶）、蛋白激酶C （PKC）和环amp依赖性蛋白激酶（PKA）的作用。在囊泡运输中对常规动力蛋白的需求将通过特异性功能阻断抗体和竞争性部分构建体进行测试。特异性肌球蛋白亚型在角膜细胞细胞膜修复中的作用将通过使用bf，通过磷酸化硫代修饰的反义寡脱氧核苷酸短暂敲除非肌肉肌球蛋白IIA和IIB的表达，并通过免疫荧光显微镜定位这些蛋白以确定其作用部位来检测。第二个有待验证的假设是，细胞膜修复的长期促进依赖于cAMP响应元件结合蛋白（CREB）介导的基因转录。为了验证这一假设，我们将在角膜细胞中稳定表达CREB的显性阴性载体，以抑制CREB介导的信号通路，并检验其对膜重封的长期促进作用。CRE- GFP （CRE- green Fluorescent Protein，绿色荧光蛋白）基因转染细胞将用于检测creb介导的基因转录并研究其受激酶的调控。为了检测PKA在受伤后向细胞核的易位，将gfp标记的PKA亚基转染到角膜细胞培养物中。DNA微阵列分析将通过识别creb依赖方式表达的基因来确定受伤后基因表达的显著变化。通过这些研究获得的知识将有助于确定在不利条件下角膜细胞膜修复的最佳条件，如干眼，长期佩戴隐形眼镜，创伤，手术和眼库中捐赠角膜的储存。

The broad long term goal of this research is to understand the basic mechanisms of cell membrane repair in corneal cells. The major hypothesis to be tested is that cell membrane repair in the cornea is dependent on vesicle trafficking, in which vesicle recruitment, transport, docking and fusion by exocytosis near the site of disruption are essential for corneal cell membrane resealing. This hypothesis will be tested by monitoring membrane resealing, exocytosis and membrane tension after micropuncture wounds. Exocytotic fusion will be disrupted by using neurotoxins to disrupt SNARE complex formation known to be required for vesicle fusion in exocytosis. Specific kinase inhibitors and activators will be used to elucidate the roles of calcium/calmodulin-dependent protein kinase (CaM kinase), protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA). The requirement for conventional kinesin in vesicle transport will be tested with specific function-blocking antibodies and competitive partial constructs. The role of specific myosin subtypes in cell membrane repair in corneal cells will be examined by the use bf by transient knockout of nonmuscle myosin IIA and IIB expression with phosphorothioate-modified antisense oligodeoxynucleotides and by the localization of these proteins by immunofluorescence microscopy to determine their site of action. A second hypothesis to be tested is that long term facilitation of cell membrane repair is dependent on cAMP Responsive Element Binding Protein (CREB) mediated gene transcription. To test this hypothesis, dominant-negative vectors of CREB will be stably expressed in corneal cells to inhibit the CREB mediated signaling pathway and examine the effect on the long term facilitation of membrane resealing. CRE- GFP (CRE-Green Fluorescent Protein) gene transfected cells will be used to detect CREB-mediated gene transcription and study its regulation by kinases. To test for the translocation of PKA to the nucleus after wounding, GFP-tagged PKA subunits will be transfected into corneal cell cultures. DNA microarray analysis will be used to determine significant changes in gene expression after wounding by identifying genes expressed in a CREB-dependent manner. The knowledge gained by these studies will help define optimal conditions for cell membrane repair in cornea under adverse conditions, such as dry eye, long-term contact lens wear, trauma, surgery and the storage of donated corneas in eye banks.