基于GWAS结果的代谢综合征"痰证"病位、病性兼杂趋势与易感基因群的相关性研究

Sputum is the basic pathological factor of the metabolic syndrome (MS). In different periods and different syndromes of the disease, the phlegm syndrome which throughout the disease course has always been different in the location and its mixed syndrome elements..For a long time, the basic researches on phlegm syndrome in MS, the law of its mixed syndromes, and the relationship between the former and congenital factor are insufficient, this affect fully play of the advantages of TCM..Therefore, based on previous work, research on phlegm syndrome is set as breakthrough point in this project. According to the latest results of the large-scale genome-wide association analysis (GWAS), the researches will be carried out such as: using syndrome elements differentiation combined with the TaqMan ? SNP genotyping technology to screen the MS phlegm susceptibility gene SNP and analyze its correlationship with phlegm syndrome; using real-time fluorescence quantitative PCR technology to find out related gene mRNA expression differences and analyze its relationship with the changing of syndrome elements and the characteristics of its mixed location syndromes..The purpose is to reveal the possible genetic basis of phlegm syndrome in different location and different mixed syndromes in MS through genetic susceptibility and gene mRNA expression levels, then to further enrich the scientific meaning of the MS Phlegm pathogenesis, provide the basis for early diagnosis, the prognosis judgment and clinical interventions of MS, offer the new ideas for researches on TCM syndrome.

痰是代谢综合征（MS）的基本病理因素，贯穿于病程的始终。研究表明，在MS的不同病理阶段、不同症候群中"痰证"的病位及兼杂证不同。长期以来，由于MS痰证特征、兼杂规律与先天因素的关系等基础研究不足，影响了中医药优势的发挥。为此，本项目拟在前期工作基础上，以MS痰证患者为对象，根据大规模的全基因组关联分析（GWAS）的最新结果，采用证素辨证结合TaqMan? SNP基因型检测技术，进行多基因、多位点的MS痰证易感基因SNP的筛选，及其与病位、病性证素的关联分析；采用实时荧光定量PCR技术，研究相关基因mRNA表达差异，分析其与证素变化、病位、病性兼杂特征的关系。目的在于从遗传易感性和相关基因mRNA 表达水平，初步揭示MS痰证易感病位以及不同病性的兼杂演变趋势可能的遗传学基础，进一步丰富MS痰证病机理论的科学内涵，为MS的早期诊断、预后判断和临床干预提供依据，也为中医证的研究提供新思路。

本项目收集MS痰证患者523例，非痰证患者352例，健康人315例。应用证素辨证方法提取各观察对象的病位、病性证素，进行证素分布情况及生化指标分析；运用SNPscanTM多重SNP分型技术，检测各基因位点基因型频率、等位基因分布及其与生化指标、痰证病位、病性兼杂的相关性；运用Real-time PCR技术检测各基因mRNA的表达水平；运用Pearson和Spearman统计学方法分析痰证素积分与理化指标，HOMA-IR与中医证素的相关性；采用社会网络分析比较痰证组和非痰证组指标的网络特征；运用Logistic回归分析与痰证相关的危险因素。结果表明，MS痰证最主要的病位在肝、脾、肾，实性病性兼杂主要涉及湿、热、气滞、血瘀，虚性病性兼杂主要涉及气虚、阴虚、阳虚、血虚；MS痰证不同症候群的共同病理特点是脾肾两虚，同时伴有一定程度的肝郁气滞，最容易兼夹湿和热，同时兼一定程度的血瘀，其夹湿和夹瘀的程度与痰的轻重有明显的关系；基因多态性分析说明痰的形成与遗传有关，同时与血脂代谢、胰岛素抵抗和炎性反应相关；MS痰证患者胰岛素抵抗与肝脾功能失调密切相关，其中实性证素主要与热密切相关，虚性证素主要与阴虚及血虚相关；社会网络分析表明，血液粘稠、血液营养物质过剩是MS痰证的主要中医病理内涵；MS痰证的独立危险因素为油腻饮食、饮酒、脑力为主劳动方式、熬夜、BMI、FPG。本研究结果既丰富了MS的病因病机理论,又从遗传学角度揭示了MS痰证的易感性。. 此外，在此基础上课题组还通过高脂饮食喂养构建脂质代谢紊乱的C57BL/6小鼠和Wistar大鼠。通过动物实验发现，临床上常用的经典方——二陈汤（化痰）、平胃散（燥湿）能改善高脂饮食诱导的小鼠和大鼠的脂代谢紊乱。二者能降低高脂饮食小鼠和大鼠体重和腹围，改善糖耐量，调节血糖和血脂代谢，其机理可能与上调CDKAL1、Caveolin-1、HNF-1β的表达有关。该研究结果从分子层面解释了机体对水谷精微运化的机理，为二陈汤和平胃散的临床应用提供实验依据，也为发挥中医药在治疗代谢性疾病中的优势奠定基础。