高濃度葡萄糖影響下，integrin β3和cytokeratin的交互作用

Abstract

血糖是提供體內細胞穩定養分的來源，因此血糖濃度受恆定機制的嚴格調控，當血糖濃度過高或過低，人體就會產生疾病。血糖失調產生的疾病中，影響最廣泛的例子是糖尿病(diabetes mellitus)。絕大多數的糖尿病患者皆死於併發症，糖尿病神經病變(diabetic neuropathy)是最常見的糖尿病併發症之一。高血糖會對神經細胞造成壓力(stress)導致糖尿病神經病變，高血糖對神經細胞產生的變異包括細胞遷移(migration)、細胞附著(adhesion)等細胞生理的改變，而影響細胞遷移和細胞附著最重要的構造是focal adhesion。由於integrin, Src, RACK是構成focal adhesion的蛋白質之一，而cytokeratin又能調節focal adhesion的功能，因此本實驗的目標是探討高濃度葡萄糖對integrin和cytokeratin的交互作用在神經細胞的影響。 本實驗以NMB7 neuroblastoma cell line為實驗對象。一開始以親和管柱捕捉和RACK1及Src有交互作用的蛋白質，質譜儀鑑定的結果發現integrin αV包含其中。接著以免疫螢光染色法證實了integrin αVβ3和focal adhesion kinase (FAK)共同組成了NMB7細胞的focal adhision。接著以Q-PCR的技術去檢測，證明K8/K18是NMB7唯一含有的cytokeratin。免疫沉澱法的結果指出integrin αVβ3及K8/K18的交互作用會隨葡萄糖濃度的增加而增加。本實驗親合管柱及質譜儀的結果除了捕捉到和葡萄糖代謝有關的蛋白質，也捕捉到了壓力反應(stress response)蛋白質，而這些壓力反應蛋白質和integrin αVβ3 complex或K8/K18有所關聯，因此在葡萄糖濃度變化下integrin αVβ3及K8/K18的交互作用或許是NMB7因應葡萄糖濃度變化所產生的壓力反應(stress response)。

The blood sugar is the stable source of energy for body's cells, so the body's homeostatic mechanism keeps blood glucose levels within a narrow range. When the blood glucose level of humans is out of this range, the individual will get diseases. One of the prevalent diseases is diabetes mellitus. Most of the patients with diabetes mellitus are died from diabetic complications. Diabetic neuropathy is one of the common diabetic complications. High blood sugar can stress nerve cells to develop the diabetic neuropathy, and also can alter cell migration and adhesion that are dependent on the dynamicity of focal adhesion in cells. Focal adhesion is composed of integrin, Src, RACK, and other proteins, and is regulated by cytokeratins. Thus, the main goal of this study is to explore the interactions between integrin and cytokeratin under high glucose situation. In this study, NMB7 neuroblastoma cell line was used as the cell model. At beginning, affinity column chromatography was used to find the proteins that interacted with RACK1 and Src. Based on the result, integrin αV was one of them through mass spectrometry. Then, the focal adhesion complex composed of integrin αVβ3 and FAK in NMB7 cells was detected by immunostaining colocalization studies. Though Q-PCR technology, K8/K18 pairings were the only cytokeratins expressed in NMB7. From the previous data, a few stress-responded proteins that interact with integrin αVβ3 complex or K8/K18 were identified. Furthermore, the interaction between integrin αVβ3 and K8/K18 dependent on glucose concentration was related to stress response.