自发性肾阳虚体质大鼠模型强化肾阳虚性状稳定传代的遗传学机制研究

The previous natural science foundation combining the innate endowment (naturally occurring aged Yang-deficiency rats during pregnancy with scared of cat, low temperature, cold food) with the acquired environment (low temperature, cold food), successfully bred yang deficiency chills unresponsive F1 kidney yang-deficiency constitution model. To optimize the above modeling method by combined with the study of anterior kidney yang deficiency constitution and DNA methylation, SNP loci association during pregnant,(epigenetic have specific effects on embryo development) and obtain breeding of 4 generations.(animal breeding principles of repeated screening of specific traits breeding 4 generations can obtain stable gene mutation) Using the modeling method by strengthening kidney yang-deficiency characters, after 4 passages, construction of varieties of spontaneous kidney yang-deficiency constitution rat model of parent,blank and child 4 generation by whole genome sequencing screening SNP locus mutation or mutation;Association-contrastive studies on sequencing screening methylation sites by DNA methylation immunoprecipitation and macroscopic characters、Viscera pathological tissue、hypothalamus-pituitary-gonad biochemical factor, differential expression gene spectrum of kidney yang deficiency obtained by previous.Around the scientific hypothesis of "stable genetic model of kidney yang deficiency should be the interaction of classical genetic traits including DNA methylation epigenetic and SNP as the core" revealing the genetic mechanism of spontaneous kidney yang-deficiency constitution model breeding passage, provides an important tool for the prevention and treatment of model of kidney yang-deficiency constitution, rich scientific connotation of kidney yang deficiency constitution.

前自然基金采用先天禀赋(自发阳虚老年鼠孕期：猫吓/低温/寒食)和后天环境(低温/寒食)造模,成功繁育阳虚畏寒反应迟钝子1代肾阳虚大鼠。结合前肾阳虚体质与DNA甲基化、SNP位点关联研究，拟优化前述造模法,采用孕期干预（表观遗传对胚胎发育有特定作用）并选育4代（动物育种原则反复筛选特定性状繁衍4代获得稳定基因突变）,逐代强化肾阳虚性状，经4次传代，构建自发性肾阳虚体质大鼠模型，对亲本、正常、子4代经WGS筛选SNP位点；MeDIP-Seq筛选DNA甲基化位点，与其宏观性状、脏器组织病理、下丘脑-垂体-靶腺轴生化因子关联比对研究，围绕“该模型肾阳虚性状稳定遗传应包括DNA甲基化的表观遗传和SNP为核心的经典遗传共同作用”的科学假说,揭示自发性肾阳虚体质模型强化肾阳虚性状稳定传代的遗传机制，为肾阳虚体质防治提供重要的模型工具和靶标，丰富肾阳虚体质科学内涵。

本研究基于中医体质病因学说，经先、后天多因素复合传代造模的选育比较，构建了肾阳虚体质大鼠模型，该模型具有肾阳虚体征明显、方法简便、成本最低，造模方法已与合作单位联合申报动物模型专利。研究中发现肾阳虚体质大鼠模型存在以下理化生物特性：（1）宏观体征：怕冷扎堆，活动度下降，不喜饮水，体重下降，便溏，两目暗淡，反应迟钝，毛色晦暗。（2）下丘脑-垂体-靶腺轴结构和功能异常：①下丘脑-垂体-靶腺轴脏器结构异常：下丘脑、垂体、肾上腺、卵巢病理切片存在结构紊乱。②下丘脑-垂体-靶腺轴功能失调：HPA轴：ACTH紊乱，CORT降低；HPT轴：T3、T4、TRH、TSHR降低，TSH升高；HPG轴：17-OHCS紊乱，LH、FSH、TSHR降低，E2、P升高。③肾主生殖功能障碍：妊娠结局不良，伴cAMP/cGMP值低下。④经右归丸治疗反证温补肾阳可改善肾阳虚体征与下丘脑-垂体-靶腺轴功能紊乱。（3）肾阳虚体质大鼠表观遗传学研究发现肾阳虚体质大鼠DNA甲基化差异位点12个，密切关联到Gdap1、Cdkal1等11个基因，基因功能富集于线粒体能量代谢、糖脂代谢以及肿瘤发生等。（4）发现肾阳虚体质大鼠latr1、lpl1、fhit3基因表达差异，与课题组前期在肾阳虚体质人群中筛选的差异表达基因一致。（5）肾阳虚体质大鼠线粒体相关研究：①肾阳虚体质大鼠线粒体DNA的SNV初筛到99个突变位点，与mt-Rnrl、Trnw等6个线粒体基因密切相关；②肾阳虚体质大鼠线粒体ATP合成减少，线粒体呼吸相关酶学活性降低，钠钾泵活性降低，初步论证肾阳虚体质存在线粒体产能障碍。综上，肾阳虚体质大鼠模型，符合中医体质学说，具有显著的肾阳虚宏观体征，存在下丘脑-垂体-靶腺轴的调控紊乱、能量代谢低下、线粒体结构和功能不良，以及DNA甲基化和线粒体基因差异，且与肾阳虚人群存在共同的差异表达基因，部分揭示了肾阳虚体质的遗传机制。