激素性股骨头坏死不同中医证型分子标志物的鉴定及其早期诊断模型的构建

Steroid-induced avascular necrosis of femoral head (SANFH) is a common orthopedic and refractory disease. Early diagnosis and treatment of SANFH is the prerequisite for its optimal efficacy. Previous studies focused on the relationship between a single predisposing gene and morbility of SANFH but not considered the gene-gene interactions which have been demonstrated to play important roles in disease progression. Thus, it is necessary to analyze the network topological features of SANFH related genes and investigate their function information in order to understand pathogenesis and progression of SANFH at a system level. Our study group have provided a novel idea of Traditional Chinese Medicine (TCM) syndrome differentiation for SANFH and also indicated their correlation with genetic variance in predisposing genes. In this study, we at first intend to detect the differential gene expression profiles in sera of SANFH patients in different TCM syndrome by gene microarray; Then, the identified genes that are closely related with the progression of SANFH may be chosen as the candidate markers; After that, their networks will be analyzed by GeneGO Meta-Core software and the hub genes may be chosen as molecular markers for SANFH in different TCM syndrome; Finally, a SANFH diagnostic classifier will be constructed by Partial Least Squares modeling based on the microarray gene expression data of the hub genes. This study may identify a list of molecular markers for SANFH in different TCM syndrome and provide a novel systems biology-based classifier that combines the differential gene expression and topological features of gene-gene interaction network which may enhance the diagnostic performance of SANFH at an early stage.

激素性股骨头坏死（SANFH）的早期诊断与早期治疗是获得最佳疗效的重要前提。以往研究多针对单个基因与SANFH易感的相关性，忽略了基因间的相互作用，不利于系统认识该病的发病机理和病程进展。本课题组前期提出股骨头坏死的三期四型中医辨证思路，并发现其中医证候与易感基因遗传变异的相关性。为确立规范统一的辩证分型标准，本课题拟检测早（以痰瘀阻络型为代表）、中（以经脉痹阻型为代表）及晚（以肝肾亏虚型为代表）期SANFH患者外周血的基因表达状况；系统分析基因表达谱与SANFH不同分型的关系，阐明病情进展中关键基因表达失衡的机制；构建SABFH相关基因的相互作用网络，筛选关键网络节点作为各分型的标志分子，并构建SANFH预测模型。本课题整合差异表达数据分析和生物分子相互作用网络分析，鉴定SANFH不同分型的标志分子，为中医辨证分型提供生物学依据，同时，也为SANFH早期诊断提供新型、高效且无创的工具。

整合激素性股骨头坏死不同中医证型患者的血清基因差异表达谱及其相关分子间的相互作用信息，筛选该病分子标志物并构建其早期诊断预测模型。方法：首先，根据课题研究方案，完成标志基因发现样本集和标志基因大规模独立验证样本集的外周血采集。其次，基于标志基因发现样本集血样数据，采用基因芯片检测技术和生物分子网络分析相整合的方法，分析不同证型与对照组外周血中基因的差异表达谱，得到激素性股骨头坏死发病相关候选标志基因；并筛选出在三个网络中处于拓扑中心位置的关键hub节点，以此为据进一步筛选痰瘀阻络证、经脉痹阻证和肝肾亏虚证的候选标志基因。采用偏最小二乘法，建立了激素性股骨头坏死发病诊断预测模型，并基于各证型候选标志基因在芯片检测样本中的表达量信息，分别建立了痰瘀阻络证、经脉痹阻证和肝肾亏虚证诊断预测模型。最后，基于标志基因大规模独立验证样本集，采用 n-倍交叉验证对上述诊断预测模型的性能进行综合评估。结果：采用基因芯片检测技术和生物分子网络分析相整合的方法，提取三种证型相关基因的交集，作为激素性股骨头坏死发病相关基因（46个），并从中筛选在疾病组显著过表达的基因作为激素性股骨头坏死候选标志基因（8个：BIRC3, CBL, CCR5, LYN, PAK1, PTEN, RAF1, TLR4）；同时筛选证型组之间显著差异基因的交集，分别获得痰瘀阻络证（4个：CD28, CD4, PLCG1, PRKCA）、经脉痹阻证（4个：PTGS2, SOS2, STAT6, TLR4）和肝肾亏虚证（5个：IFIT1, IRF7, ISG15, MAPK14, RHOU）的候选标志基因；采用偏最小二乘法，分别建立激素性股骨头坏死发病预测模型，痰瘀阻络证、经脉痹阻证和肝肾亏虚证辨证诊断预测模型；采用十倍交叉验证方法，计算得到各模型的预测性能稳定且良好，其中，激素性股骨头坏死的发病诊断预测模型性能最优，准确率达到90%以上，三个辨证模型的预测性能也较好，准确性均在72%以上。结论：股骨头坏死的早期诊断预测模型，为激素性股骨头坏死的早期诊断提供新型、高效且无创的工具。