基于microRNAs调控的慢性乙型肝炎脾胃湿热证发生的分子机制研究

Chronic Hepatitis B(CHB)is the critical infectious desease which endangered chinese people's health heavily. Clearing the virus of CHB is the difficulty for western medicine.Syndrome differentiation lacks objective diagnostic code,which restricts the promotion of the experiences of Traditional Chinese Medicine(TCM) experiences and clinical level on CHB.The study of objectification and standardization of TCM syndrome is the key to prevent and treat chronic hepatitis B .Based on the National Key Technology R&D Progranm in the 11th Five Year Plan of Chinaprevious, our teams found that the damp-heat in the spleen-stomach syndrome is the most common syndrome in CHB(mild),and we screened out the 9 significant difference genes and 168 significant difference proteins of the most common sydrome with the help of the tanscriptomics and proteomics technology,but the both results are not entirely consistent,so we infer that it is caused by the post-transcriptional control.MicroRNAs is a research hotspot in recent years，its integrity, diversity and complexity are similar to the characteristics of Chinese medicine.We first introduce miRNAs to the project，We found differences in serum microRNAs between spleen stomach damp heat syndrome and the asymptomatic with chronic hepatitis B by using microarray and bioinformatics technology，then we use RT-PCR, classical prescriptions to validation of microarray results, we finally revealed the molecular mechanism of chronic hepatitis B with spleen stomach damp heat syndrome and the role of cleaning away heat and dampness,and we try to find molecular markers ( spectrum ) of spleen stomach damp heat syndrome in post transcriptional level.

慢性乙型肝炎（慢乙肝）严重危害我国人民健康，目前西医治疗难以彻底清除病毒。中医辨证缺乏客观化诊断标准限制中医治疗慢乙肝宝贵经验推广和临床水平提高。中医证候客观化、标准化研究是提高中医防治慢乙肝水平的关键。我们在国家十一五科技重大专项（2009ZX10005-020）研究中借助转录组学和蛋白组学等方法筛选出慢乙肝脾胃湿热证9条共表达差异显著性基因和168个差异表达蛋白，但二者解释不完全一致，推测可能存在转录后等水平调控。近年研究热点microRNAs调控的整体性、多层次性及复杂性与中医学特点相似。本项目首次引入microRNAs，先借助微阵列芯片及生物信息学等技术手段找到慢乙肝脾胃湿热证和无症状者血清差异microRNAs，再用RT-PCR、经典方药等验证芯片结果，最终揭示慢乙肝脾胃湿热证发生的分子机制及清热化湿法的作用分子机制，并试图在转录后等水平找到脾胃湿热证分子标志物（谱）。

我国属于慢性乙型肝炎(Chronic hepatitis B, CHB)高流行区, CHB是我国重大传染病之一。轻度CHB脾胃湿热证的差异表达microRNAs共60条(P<0.05), 26条上调, 34条下调; 2倍以上差异表达的microRNAs 23条, 上调10条, 其靶基因共500个, 通过GO分析其功能主要涉及转录因子活性、免疫系统的发育、转运活动、细胞粘附等生命过程; 下调13条, 其靶基因共499个, 通过GO分析其功能主要涉及转录因子活性、转移酶的活性、核酸结合、蛋白结合、转运、免疫系统的发育等生命过程.可见轻度CHB脾胃湿热证存在特异性差异microRNAs表达谱, 涉及多个生命过程。这为乙肝中医证候诊断客观化提供依据，有利于提高中医药防治CHB临床疗效. HBV慢性感染受特异性microRNAs调控，差异表达microRNAs共69条（P<0.05），28条上调，41条下调；GO分析及Pathway分析得到其功能主要涉及生物粘附、转录正/负调控、生物合成过程的正/负调控、氮化合物的代谢过程的正/负调控、蛋白质定位、蛋白氨基酸的磷酸化、Notch信号传导途径、细胞凋亡、Wnt信号通路、Hedgehog信号通路、T细胞受体信号通路、MAPK信号通路、TGF-β信号通路、B细胞受体信号通路、ErbB信号通路、p53信号通路等。这揭示慢性HBV感染的分子机制，更新了目前的HBV致病机制。轻度慢乙肝/慢性HBV携带者两组间差异表达microRNAs共65条（P＜0.05），38条上调，27条下调；GO分析及Pathway分析得到其功能主要涉及细胞增殖、生物黏附、生物合成过程的正/负调控、大分子生物合成过程中的正/负调控、蛋白氨基酸的磷酸化、RNA的生物合成过程、Wnt信号通路、MAPK MAPK信号通路、Notch信号传导途径、Hedgehog信号通路、T细胞受体信号通路、TGF-β信号通路、mTOR信号通路、趋化因子信号通路JAK-STAT信号通路、钙离子信号通路等。结论：轻度慢性乙型肝炎炎症活动受特异性microRNAs调控，其发生的机制涉及多个生命过程及通路.