miRNA在慢性再生障碍性贫血发病相关机制及与中医辨证分型相关性的研究

Recent studies show that MicroRNA is involved in hematopoietic process and regulate hematopoietic stem cell differentiation. Also, MicroRNA plays an important role in immune system. In the previous research, we found that the pathogenesis of CAA is associated with hematopoietic stem cell defect and abnormal immune system, moreover, TCM syndrome differentiation types also have more relationship with the above changes. We presume that hematopoietic stem cell defect and abnormal immune function may be caused by the abnormal patterns of MiRNA and its target genes abnormal expression, also, there are some differences of MiRNA patterns among different syndrome differentiation types which can lead to different therapeutic results. On the basis of above hypothesis, we design this study. MiRNA microarray and real-time PCR are used to investigate CAA specific miRNA, and miRNA related target genes are predicted by using bioinformatics method. Combined with detection of other immune indexes, we will discuss the new pathogenesis of CAA, analyze the substantial bases of syndrome differentiation type in CAA, explain kidney deficiency is the main Chinese medicine pathogenesis of CAA. These results will supply theoritical base and experimental data in CAA treatment and subjective study of syndrome.

近年来研究发现微RNA（microRNA,MiRNA）参与造血过程，在造血干细胞分化过程中发挥调控作用。而且MiRNA对免疫系统亦有一定的调节作用。我们在前期国家自然科学基金资助的研究中发现慢性再生障碍性贫血（CAA）与造血干/祖细胞缺陷和免疫异常相关，CAA中医不同证型之间存在造血干/祖细胞数量和免疫异常方面的显著差异。我们推测造血干/祖细胞缺陷和免疫异常与MiRNA及其靶基因异常表达有关，而且CAA不同证型间存在MiRNA表达的差异，从而导致治疗的难易和预后的不同。基于上述假说，我们设计了本课题。拟采用基因芯片结合实时荧光PCR法筛选CAA特意性MiRNA，利用生物信息学方法推测其靶基因，力求阐释CAA新的发病机制。同时结合与造血以及免疫相关的指标，全面综合分析CAA辨证分型的物质基础，探讨CAA肾虚病机的本质，为证的客观化研究以及CAA的中医药治疗提供理论依据和实验数据。

慢性再障的发病机制至今尚未明确。祖国医学在慢性再障的辨证论治方面有其优势，对慢性再障辨证分型的物质基础研究也已发展到瓶颈阶段。随着微小RNA的发现，证实许多疾病的深层次发病机制均有miRNA的参与。本研究内容分为两部分：第一部分是慢性再障患者miRNA的筛选和验证以及作用靶基因推测，分析慢性再障不同证型之间miRNA表达的差异性。第二部分是围绕慢性再障造血干/祖细胞缺陷和免疫异常的发病机制，探讨miRNA的异常表达与其相关性。结果发现，慢性再障患者外周血和骨髓与正常人相比有差异性表达的microRNA分别是66个和57个，经RT-PCR验证后与辨证分型相关的分别有3个和2个。外周血中miR-155表达上调，miR-126和miR-199表达下调。肾阴阳两虚组和肾阴虚组miR-155上调的倍数最高，肾阳虚组最低，有统计学差异。对于miR-126，肾阳虚组、肾阴阳两虚组和肾阴虚组三组之间存在统计学差异，肾阴虚组表达最低，其次是肾阴阳两虚组，肾阳虚组最高。对于miR-199，肾阴阳两虚组和肾阴虚组表达最低，肾阳虚组最高，有统计学差异。慢性再障患者骨髓中miR-96表达上调，miR-133表达下调。肾阴阳两虚和肾阴虚组miR-96表达倍数最高，肾阳虚组最低，有统计学差异。对于miR-133，肾阳虚组、肾阴阳两虚组和肾阴虚组三组之间存在统计学差异，肾阴虚组表达最低，其次是肾阴阳两虚组，肾阳虚组最高。生物学信息分析发现，上述差异表达的microRNAs的预测靶基因与红细胞调控、免疫调节以及凋亡相关。免疫机制研究发现，EPO、SCF、IFN-γ、TNF-α、Fas抗原阳性率和凋亡率均以肾阴虚组为严重，肾阳虚组最轻，而miR-155与IFN-γ、TNF-α过度分泌相关，miR-96与Fas抗原阳性率和凋亡率相关。提示慢性再障中医辨证分型与特异性microRNAs的表达相关。综合上述结果，从miRNA角度研究慢性再障的发病机制不仅有助于从基因水平揭示其发病机制，而且对进一步阐明慢性再障辨证分型的物质基础，阐释“阳虚易治，阴虚难调”的中医理论，丰富慢性再障的诊疗体系具有重大意义。项目资助发表论文9篇，其中SCI论文1篇。培养硕士研究生1名，已取得硕士学位。