高血糖致气道机械防御屏障受损的分子介导机制

Pulmonary infection commonly occures in patients with diabetes mellitus due to chronic hyperglycemia stimulation. Airway mechanical defence barrier manily consists of two parts,the integrity of airway epithelial and effective mucociliary clearence. The mechanisms underlying hyperglycemia induces damage of airway defence barrier is still unknown. Gap junction protein connexin43(Cx43) is tightly related to the process of hyperglycemia-induced dysfunction in many organs, and so we assume that it may be invovled in the process of hyperglycemia-induced pulmonary infection. In the present study, the expression vectors of mutated Cx43 gene were constructed and transfected into airway epithelial cells, and then glucose was added to stimulate the cells.The changes of the expression and the location of Cx43 will be observed to identify the target sites of Cx43 which are invovled in hyperglycemia-destructed airway defence barrier.To identify the molecular structural principle of hyperglycemia-damaged airway integrity, The effects of hyperglycemia on the structural proteins of airway epithelial will be detected by immunoprecipitation,X-ray crystal diffraction, patch clamp technique and so on.We will utilize microinjection Fluorescence tracing assay,flow cytometry,radiochemical-binding assay,Fluorescence Polarization and Fluorescent microsphere imaging method to make clear the status of airway mucociliary clearence and which signal factors are invovled in this process.To identify the molecular conformation,signal factors and biological dynamics process of hyperglycemia-impiared airway defence function,it will provide us useful enlightenment for how to prevent and control hyperglycemia-induced pulmonary infection in clinical work.

糖尿病患者长期的高血糖状态易致肺部感染，其致死率极高。气道机械防御屏障主要由气道上皮完整性及有效的粘液纤毛清除功能组成，高血糖损害气道防御屏障致肺部感染的机制目前尚无报道。缝隙连接蛋白Cx43参与高血糖致各靶器官功能障碍的过程，推测其可能介导高血糖致肺部感染。本研究构建Cx43基因突变表达载体，转染人气道上皮细胞，予以葡萄糖刺激，观察Cx43表达及定位变化，探讨Cx43参与高血糖破坏气道防御屏障的可能关键靶位点。利用免疫共沉淀法、X-线晶体衍射技术及膜片钳技术等检测高血糖对气道上皮各结构蛋白表达及定位的影响，明确Cx43参与高血糖破坏气道上皮完整性的分子结构原理；同时采用微注射荧光示踪分子法、流式细胞术、放射化学结合法、荧光偏振技术及荧光微球体显像法等检测气道粘液纤毛功能受抑情况，探讨参与其中的信号因子。明确介导其中的分子构象、信号链接及生物动力学过程对高血糖致肺部感染的防控有重要启示。

一、.项目背景：.肺部感染是糖尿病患者的常见并发症，且为常见致死病因。气道上皮细胞层结构的完整性是气道防御屏障的关键，一旦上皮完整性破坏，气道机械防御屏障结构性和功能性破坏，从而导致气道炎症或感染的发生。Cx43是Cx家族最主要的成员，连接蛋白可与紧密连接、粘附连接及细胞骨架蛋白等结构发生作用以维护气道上皮结构的完整性。高血糖可能引起Cx43的高磷酸化及表达量的变化，致组织及结构的完整性受损或消失及各靶器官功能障碍。.二、.主要研究内容：.1. 高血糖刺激对Cx43在人气道上皮细胞的表达定位的影响研究：各Cx43磷酸化位点突变基因表达载体的酶切鉴定及转染；实时荧光定量PCR及Western blot法检测Cx43的表达水平及蛋白磷酸化水平，明确高血糖对Cx43的表达的影响，分析高血糖致Cx43磷酸化水平的变化，推测可能作用的具体磷酸化位点。.2. Cx43参与高血糖致气道上皮完整性受损的研究：包括使用Western blot法、免疫共沉淀、激光共聚焦等方法Cx43对气道上皮结构蛋白如ZO-1、E-cadherin等蛋白表达定位及蛋白间相互作用的影响；通过经上皮电阻测量法（TER）、气道上皮通透性、GJIC等检测了解气道上皮完整性，明确Cx43对气道上皮完整性的影响。.三、 重要结果：.1..在高糖致气道上皮结构受损过程中，Cx43的磷酸化可影响Cx43在细胞内的表达，具有关键的调节作用。.2..在高糖刺激气道上皮细胞以后，Cx43的磷酸化可引起紧密连接及粘附连接蛋白表达量的降低，致气道上皮结构受损。.3..高糖可致气道上皮完整性受损，而Cx43的高磷酸化为参与上述过程的重要调节因素。.四、关键数据：.1..成功构建Cx43磷酸化突变载体pEGFP-N1-Cx43279-MU、pEGFP-N1-Cx43365-MU和pEGFP-N1-Cx43368-MU。.2..萄糖浓度的逐渐增加，Cx43 mRNA及蛋白表达明显降低。葡萄糖刺激可增加Cx43磷酸化蛋白表达及MAPK、 Scr及PKC的磷酸化水平。而三种激酶抑制剂均可下调高糖刺激引起的Cx43高磷酸化水平。.3..高糖刺激可明显减少Cx43的mRNA和蛋白的表达及ZO-1及E-cadherin，增加pCx43的水平，而转染Cx43磷酸化突变体后可抑制高糖对上述蛋白表达的作用。.4..高糖刺激可明显降低气道上