Mechanisms of cell membrane repair in corneal cells

这项研究的长期目标是了解角膜细胞膜修复的基本机制。需要检验的主要假设是，角膜细胞膜的修复依赖于囊泡的运输，其中囊泡在破裂部位附近通过胞吐的募集、运输、对接和融合是角膜细胞膜重新密封所必需的。这一假说将通过监测微穿刺伤后的膜重新封闭、胞吐和膜张力来验证。胞吐融合将通过使用神经毒素来扰乱已知的胞吐中囊泡融合所需的圈套复合体的形成。具体的激酶抑制剂和激活剂将被用来阐明钙/钙调蛋白依赖的蛋白激酶(CaM)、蛋白激酶C(PKC)和环磷酸腺苷依赖的蛋白激酶(PKA)的作用。将用特定的功能阻断抗体和竞争性部分结构来测试在囊泡运输中对传统动蛋白的需求。特定的肌球蛋白亚型在角膜细胞膜修复中的作用将通过用硫代修饰的反义寡核苷酸瞬时敲除非肌肉肌球蛋白IIA和IIB表达的bf以及通过免疫荧光显微镜定位这些蛋白来确定它们的作用部位来检验。需要检验的第二个假设是，细胞膜修复的长期促进依赖于cAMP反应元件结合蛋白(CREB)介导的基因转录。为了验证这一假说，CREB的显性-负性载体将在角膜细胞中稳定表达，以抑制CREB介导的信号通路，并检测其对膜再封闭的长期促进作用。将Cre-GFP(Cre-Green Follow Protein)基因导入细胞，用于检测CREB介导的基因转录，并研究其在蛋白水解酶中的调控作用。为了检测创伤后PKA向细胞核的移位，GFP标记的PKA亚单位将被导入角膜细胞培养中。DNA微阵列分析将用于确定创伤后基因表达的显著变化，方法是识别以CREB依赖的方式表达的基因。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，例如干眼、长期配戴隐形眼镜、创伤、手术和将捐赠的角膜储存在眼库中。

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.