基于microRNA分子检测的轻微型肝性脑病的快速准确诊断方法的研究

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome associated with advanced liver diseases and/or portosystemic shunting. Minimal HE (MHE) is the mildest stage of HE without clinical evidence of mental change. The early diagnosis of MHE is the key to stop patients from developing overt HE; which threatens their lives. In view of the remarkable disadvantages of traditional diagnosis methods of MHE such as time-consuming, low accuracy and susceptibility to external factors, we aim to develop a novel, rapid, efficient and accurate method for MHE diagnosis based on microRNA detection. Firstly, a group of miRNAs will be selected as the candidate biomarkers for the diagnosis of MHE. Then the serum samples from cirrhotic patients with or without MHE will be subjected to abundance assessment of candidate miRNAs by real-time PCR. And the biomarker miRNAs will be determined by comparing miRNA abundances of the above two groups. Finally, the rapid and efficient diagnosis method based on the biomarker miRNAs will be established by combining functional magnetic nanoparticles and chemiluminescence. The novel technique is believed to set up an efficient diagnosis system of MHE, which is promising to be widely used in clinic. Moreover, the study is supposed to provide critical information to the investigation of pathogenesis of HE.

肝性脑病是一种由于肝功能严重障碍和/或门-体分流异常所导致的神经功能障碍。轻微型肝性脑病是肝性脑病最初的阶段，无明显临床症状，该状态的及时诊断是阻止其进一步发展成威胁病人生命的显性肝性脑病的关键步骤。为了突破轻微型肝性脑病传统诊断方法存在的耗时漫长、准确率低、易受外界因素影响等明显缺陷，本课题拟发展一种基于microRNA分子检测的轻微型肝性脑病的诊断新方法。首先根据先前的研究成果和文献调研，构建该疾病诊断的标记microRNA候选库，然后通过实时荧光定量PCR实现轻微型肝性脑病临床样本的候选库microRNA的定量检测，并确定该疾病的标记microRNA。最后建立基于功能磁性纳米颗粒和化学发光的快速高效检测上述标记microRNA的方法，并通过临床样本的检测来验证方法的有效性。该新技术的研究有望形成一套完善的诊断体系，突破轻微型肝性脑病诊断甚至是机制研究的瓶颈，满足临床诊断的迫切需求。

肝性脑病是一种由于肝功能严重障碍和/或门静脉－体循环分流异常所导致的神经功能障碍，轻微型肝性脑病是肝性脑病最初的状态。microRNA的分子标记作用在轻微型肝性脑病的早期诊断中具有至关重要的作用，可以防止其进一步发展产生不可逆的损伤。本项目计划研究轻微型肝性脑病中有分子标记意义的microRNA及其潜在机制，主要研究内容包括：1）构建轻微型肝性脑病诊断的标记microRNA候选库；2）轻微型肝性脑病临床样本的候选库miRNA的定量分析并确定该疾病的标记miRNA；3）基于标记miRNA的快速检测方法的建立与方法验证。首先，项目通过大量文献调研建立了一个包含100个轻微型肝性脑病诊断的潜在标记microRNA的侯选库，进而对侯选库中一些microRNA在轻微型肝性脑病发病机制中的具体作用进行了深入研究，不仅在轻微型肝性脑病发病机制方面获得了突破性的发现，还为这些microRNA作为标记分子提供了坚实的理论依据，为最终具有临床意义的标记microRNA的确定大大缩小了范围。然而，除了具有分子标记microRNA的确定，高效检测microRNA的方法研发也是实现其应用的必需工具。本项目通过合成和优化磁性纳米颗粒，研发了一种全新的高效检测microRNA的方法，在核酸检测方面实现了明显突破，有望成为microRNA水平检测的有力工具。由于脑组织的特殊性，microRNA作为标记分子在各种疾病尤其是神经性疾病的早期诊断和干预治疗方面具有广阔的应用前景。