Pepsin as biomarkers of reflux

胃蛋白酶作为生物标志物胃液间歇性反流进入食管和食管外是正常的生理事件。因此，组织如食管、喉、鼻咽和气道必须具有适当的粘膜保护机制。目前尝试使用侵入性诊断工具（例如24小时pH监测或阻抗测量）来测量人体中的反流，这两种技术都涉及将薄壁管穿过食道放置到食管中约45 cm的距离。因此，为了确定反流水平，需要鉴定和定量生物标志物。胃蛋白酶是胃液中的主要酶成分（以0.2- 1 mg/ml存在），是此类生物标志物。本项目的目的有两个方面：（1）确定胃蛋白酶的正常反流水平，并开发一个简单的测定系统，可以商业化开发。(2)以增强组织特异性保护机制。目前，没有商业测定可用于测量生物液体和组织中的胃蛋白酶，本研究的中心部分将是与纽卡斯尔大学和技术学院的Pearson教授一起开发这种测定，该技术学院是一家位于东约克郡船体上的金斯顿的SME。迄今为止，文献中几乎没有或没有信息来定义这些组织中存在的反流或保护机制的正常水平。粘液，碳酸氢盐，碳酸酐酶同工酶，应激蛋白和细胞粘附分子的差异表达都有牵连。在纽卡斯尔大学的ICaMB中，学生将使用体外细胞和外植体培养技术来研究胃蛋白酶对食管、喉、鼻咽和气道粘膜完整性的影响。所采用的技术将包括免疫组织化学、定量PCR、ELISA和多重技术，以测量粘蛋白、碳酸酐酶同工酶、上皮应激蛋白和细胞粘附分子。在Technostics，学生将接受商业检测开发的各个方面的培训，包括使用计算机程序进行抗体设计以识别胃蛋白酶蛋白内的关键表位;单克隆抗体生产;侧流试纸条技术。将在Technostics和纽卡斯尔大学进行试验开发，包括评估临床环境中的重现性、可靠性和易用性。这将涉及学生与工业和临床同事的互动。因此，学生将在商业和学术环境中接触到广泛的技术。该胃蛋白酶检测试剂盒在完全开发和验证后，将通过受试者咳出的痰液中胃蛋白酶的出现来识别反流事件，并且可以在医生的手术中使用试纸形式。它还将在疾病诊断和指导治疗管理中应用，其中过度反流可导致呼吸消化道中的组织损伤。最近发现的与反流相关的健康问题充分证明了这种测定的必要性。我们和其他人已经证明，胃液反流与几种上呼吸道疾病的发展有关，例如鼻炎、鼻窦炎、渗出性中耳炎、语音障碍、声门癌和喉癌以及几种肺部疾病，例如哮喘和COPD。我们最近证实了反流在肺移植排斥反应中的关键作用。因此，了解反流的性质和组织如何保护自己是关键问题。此外，医疗实践迫切需要一种简单，易于使用的反流诊断工具。

Pepsins as biomarkers Intermittent reflux of gastric juice into the oesophagus and beyond is a normal physiological event. Consequently tissues such as the oesophagus, larynx, nasopharynx and airways must have mechanisms of mucosal protection in place. Currently attempts to measure reflux in humans use invasive diagnostic tools such as 24 hour pH monitoring or impedance measurement, both techniques involve placing a thin walled tube through the nostril into the oesophagus for a distance of about 45 cm. Therefore in order to determine reflux levels a biomarker needs to be identified and quantified. Pepsins, a major enzyme component of gastric juice (present at 0.2-1mg/ml) are such biomarkers. The aims of this project are two fold; (1) To define the normal levels of reflux and develop a simple assay system for pepsin that can be developed commercially. (2) To characterise tissue specific protection mechanisms. At present no commercial assay is available to measure pepsin in biological fluids and tissues and a central part of this study will be to develop such an assay with Prof Pearson at Newcastle University and Technostics, an SME based in Kingston upon Hull, East Yorkshire. To date there is little or no information in the literature to define the normal levels of reflux or the protective mechanisms present in these tissues. Mucus, bicarbonate, differential expression of carbonic anhydrase isoenzymes, stress proteins and cell adhesion molecules have all been implicated. In ICaMB at Newcastle University the student will use in vitro cell and explant culture techniques to investigate the effects of pepsin on mucosal integrity in the oesophagus, larynx, nasopharynx and the airways. Techniques employed will include immunohistochemistry, quantitative PCR, ELISA and multiplex techniques to measure mucins, carbonic anhydrase isoenzymes, epithelial stress proteins and cell adhesion molecules. While at Technostics the student will be trained in all aspects of commercial assay development including antibody design using computer programs to identify key epitopes within the pepsin protein; monoclonal antibody production; lateral flow dipstick technology. Assay development including assessing reproducibility, reliability and ease of use in the clinical setting will be carried out at both Technostics and Newcastle University. This will involve the student interacting with industrial and clinical colleagues. Thus the student will be exposed to a wide range of techniques in commercial and academic environments. This pepsin assay when fully developed and validated will identify reflux events by the appearance of pepsin in sputum expectorated by the subject and in a dip stick format could be used in the doctors surgery. It will also have applications in disease diagnosis and directing treatment management, where excessive reflux can lead to tissue damage in the aero-digestive tract. The need for such an assay is well demonstrated by the recent identification of health problems associated with reflux. We and others have demonstrated reflux of gastric juice is implicated in the development of several upper airway diseases e.g. rhinitis, sinusitis, otitis media with effusion, voice disorders, globus and laryngeal cancer and several lung diseases e.g. asthma and COPD. We have recently demonstrated a key role for reflux in lung transplant rejection. Consequently understanding the nature of the refluxate and how tissues defend themselves are critical questions. Also medical practise is crying out for a simple, easy to use diagnostic tool for reflux.