外切酶体微小RNA调控乙肝肝纤维化的机制研究及其临床应用

The principal cells responsible for promoting the accumulation of extracellular matrix proteins during hepatic fibrogenesis are activated hepatic stellate cells (HSC). The study of regulation mechanism of HSC activation has its profound clinical meanings. Individual microRNA (miRNA) can targetnumerous mRNAs and have been referred to as the "micromanagers" of cellular gene expression, they can orchestrate gene expression profiles associated with phenotypic changes in cells and disease progression. They might evolve as biomarkers in the diagnosis of various diseases and also might represent targets for novel therapeutic strategies against different disease.Exosomes are cell-derived extracellula rvesicles that promote cell-cell communication and immunoregulatory functions. These "bioactive vesicles" shuttle various molecules, including miRNAs, to recipient cells. Inappropriate release of miRNAs from exosomes may cause significant alterations in biological pathways that affect disease development, supporting the concept that miRNA-containing exosomes could serve as targeted therapies for particular diseases. In this study, we focus on using what is learned in pre-clinical studies to do smarter things in the course of predicting, preventing, diagnosing, and treating diseases, which meets the need of translational medicine. Firstly, we will systematically analyze the differently expression profile of miRNA in LX-2 cell lines and liver firosis patients by microarray analysis, which would revealed a panel of miRNA that were specifically regulated during the process of liver fibrosis. Secondly, the level of regulated miRNA will be detected in clinical samples (liver tissue and plasma) with diverse technologies to confirm the result from microarray, which has innovative characteristic. In term of liver fibrogenesis, our study could lead to a further comprehension of liver fibrosis regulation mechanism, also provide the clue to develop future therapeutic candidates to prevent or treat hepatic fibrosis. Finally, by retrospective analysis, we would construct a risk predicting model for liver fibrosis, also to help physician to make a optimal treatment for HBV-infected patients with ALT ≤ 2 ULN, which has a great clinical significance.

肝星状细胞(HSC)的激活导致肝纤维化发生，对其调控机制的研究有深刻的临床意义。微小RNA（miRNA）调控多种基因的表达，有可能成为新的诊断生物学标记和治疗靶标，循环miRNA大部分存在于外切酶体中，并通过外切酶体在细胞之间传递从而发挥的作用。本项目从临床问题出发，通过机制研究之后指导临床实践，符合转化医学思维。以exosome及miRNA为切入点，通过生物芯片技术筛选肝纤维化过程中差异表达的miRNA谱，并在临床病例中验证，源头创新性强，同时使用细胞系进行功能研究，寻找差异表达miRNA调节的靶位点及其参与的信号通路，加深对于肝纤维化发生、发展及调控系统的理解，为寻找新的抗纤维化治疗靶点提供线索。通过回顾性分析，结合临床资料、基因多态性及差异表达miRNA谱，构建肝纤维化风险预测模型，并且通过前瞻性研究指导肝功能正常或轻度异常的慢乙肝患者的抗病毒治疗，具有实际的临床应用价值。

肝纤维化是各种肝脏疾病发展的最终共同途径，肝纤维化的早期诊断可用于指导肝纤维化高风险患者的早期治疗，还可以避免对肝纤维化低风险患者的过度治疗，从而进一步提高治疗有效率，减少医疗资源的浪费，降低医患纠纷。本项目通过1年的研究，在基础研究方面，通过小鼠四氯化碳肝纤维化模型，发现miR-22 可以直接作用于骨形态发生蛋白(bone morphogenetic proteins，BMP)7的 UTR 抑制其表达，从而促进肝纤维化过程。肝纤维化进展相关SNP研究结果显示TGF-β1 (CC型)和TIMP-2 (GG型)与肝纤维化风险相关，而MMP-3与DDX5 SNP与肝纤维化无关。临床研究方面分析了FibroScan检测肝纤维化的失败因素，结果显示FibroScan总检测失败率为3.34%，其中2.49%为不可信检测，0.85%为不成功检测，logistic回归分析显示女性、年龄大于50岁、肥胖、腹水为检测失败的独立预测因素。通过以上研究，加强了对肝纤维化发生机制的了解，明确下一步miRNA靶位点，为肝纤维化的早期诊断、未来的新药研发、基因治疗策略的制定提供理论依据。