血瘀体质型非创伤性股骨头坏死相关microRNA组学分析及其靶基因调控网络研究

NONFH is a complicated disease with multi-factor, multi-gene and multi-channel and its pathogenesis involves different levels of biological regulatory networks. Constitution factor of TCM play an important role in the formation, development and outcome of NONFH. Previous results of nature project findings show that blood stasis constitution is one of high-risk constitution for NONFH. New study findings have shown that miRNAs are an important regulatory factor of osteonecrosis and the increasing or decreasing of their expression levels can lead to serious pathological consequences. Plasma miRNA has features with a content-rich, stable and easy to be measured and can be used as a new class of biomarkers for many diseases in the early screening, diagnosis and prognosis assessment study. To take cold physique as reference, this study will select typical NONFH cases with blood stasis type by microfluidic miRNA expression microarray, bioinformatics analysis, qRT-PCR and western blot. This study intends to screen and verify related microRNA genomics and its target genes in the NONFH, to draw the regulatory network of miRNA and target genes, to preliminarily discuss molecular mechanism of related miRNA genomics, and to explore scientific connotation of disease combined with the corresponding constitution factor of TCM. The results could provide new ideas and treatment targets for diagnosis and treatment NONFH by TCM constitution differentiation.

非创伤性股骨头坏死(NONFH)是多因素、多基因、多通路的复杂疾病，其发病机理涉及不同层次生物网络调控。前期自然基金项目研究表明，中医体质因素在NONFH的形成、发展和转归中发挥重要作用，血瘀体质是NONFH的高发体质之一。新近研究认为，miRNA是骨坏死重要调控因子之一，其表达量的异常可引发病理改变，血浆miRNA具有含量丰富、性质稳定、易于检测等特征，已作为一类新型生物标志物用于多种疾病的早期筛查、诊断和预后评估研究。本研究拟选择血瘀体质型NONFH典型病例和阳虚体质型作对照，利用微流体miRNA表达谱芯片、生物信息学分析、qRT-PCR和western blot等技术，筛选并验证血瘀体质型NONFH相关miRNA及其靶基因，绘制相关miRNA与靶基因的调控网络，初步探讨miRNA在NONFH发病中的分子机制，阐述NONFH体病结合理论的科学内涵，为辨体诊治NONFH提供新思路和新靶标。

目的：通过NONFH的流行病学调查，进一步明确其高发体质类型及高发因素，并通过检测NONFH患者外周血中差异表达的miRNA，建立miRNA表达谱，初步探讨miRNA在NONFH发病中的分子机制，为NONFH的早期诊断和治疗提供新的思路与理论依据。.方法：签署知情同意书后收集2015年1月至2018年12月在甘肃省中医院骨科就诊的NONFH患者共200例，建立NONFH数据库，并对疾病发病人群、高发体质类型等进行统计学分析。采集患者及健康志愿者血液标本，提取血浆标本并保存于-80℃冰箱。采用RT-PCR法检测其血浆miRNA相对表达量，筛选差异表达的miRNA。应用实时荧光定量PCR法对差异表达的miRNA进行验证。采用TargetScan、microRNAorg及PITA三个在线数据库共同对差异miRNA进行靶基因预测，并对靶基因进行生物信息学分析。.结果：甘肃地区NONFH患者流行病学调查研究发现，不同性别、年龄患者的中医体质分布差异均无统计学意义，不同病程患者的中医体质分布差异有统计学意义；在AIONFH患者中男46例(92.0%)，女4例(8.0%)；中医体质中痰湿质频次31，占48.4%、平和质频次12，占18.7%、阳虚质频次7，占10.9%、湿热质频次6，占9.4%；在对AIONFH的CYP2C8基因rs17110453位点多态性分析中，未发现CYP2C8基因rs17110453位点多态性与AIONFH明确关系。对于血瘀质股骨头坏死差异miRNA筛选研究中，最终确定血瘀质NONFH差异表达miRNA有5个，三个在线数据库共筛选出交集靶基因11个，仅表达下调miRNA中hsa-miR-365a-3p检出交集靶基因11个，余未检测出交集靶基因。GO分析显示差异表达miRNA主要与受体结合、蛋白与酶的活性有关。Pathway分析显示，血瘀质NONFH差异表达miRNA靶基因主要富集于轴突导向、Rap1、甘油磷脂代谢、T细胞受体、血小板活化、成骨细胞分化等多个信号通路。.结论：进一步明确了NONFH的高发体质类型，发现痰湿质是AIONFH的高发中医体质，完善了NONFH病例数据库，获得了血瘀质NONFH差异表达miRNA，这些miRNA可能通过调控信号通路的传导参与神经发育、信号转导、解剖结构形态、细胞代谢过程及解剖结构的发展，对疾病的发生发展产生影响。探讨m