节点文献
基于“肾为先天之本”2型糖尿病肾虚证的miRNA研究
The Research of MiRNA Expression of Kidney Deficiency Syndrome of T2DM Based on the "The Kidney Is the Congenital Foundation" Theory
【作者】 孙玉文；
【导师】 王米渠；
【作者基本信息】 成都中医药大学 ， 中西医结合基础， 2013， 博士
【摘要】 目的：糖尿病已经成为威胁人民健康的重要非传染疾病之一,在我国尤其以2型糖尿病为主。2型糖尿病的发病既有复杂的遗传背景,同时与环境等因素密切相关。以2型糖尿病为载体,从中医“肾为先天之本”理论为基础,能够更好的阐述“遗传-环境-疾病”相互作用和中医的整体观。本文在前期全基因组表达谱研究平台上,引入miRNA芯片技术,旨在探索2型糖尿病肾虚证的表观遗传学机制。1.通过对中医古籍中消渴病的梳理和对现在表观遗传学方法的探讨,从理论上讨论将表观遗传学方法引入到中医藏象学的研究的创新和应用价值。2.通过对793例成都地区2型糖尿病患者的家族史回顾性调查,揭示遗传因素对肾虚证的影响。3.从2型糖尿病患者中筛选出8例典型肾虚证和非肾虚证患者,并分别和4例正常人对比,分析差异表达miRNA并筛选意义较大的miRNA进行PCR验证,从分子水平初步探讨肾虚证糖尿病的分子机制变化。方法1.2型糖尿病人群的病证结合研究：采用肾虚证辨证因子及等级量化表对2型糖尿病人群面对面进行病证结合调查,采用统计学分析方法,探讨分析肾虚证糖尿病人群的分布特征。2. miRNA表达谱芯片异常表达基因的筛选：采用miRNA表达谱芯片技术,对筛选的12例样本分3组两两对比,进行miRNA基因表达谱试验,对差异miRNA进行靶基因预测。3.对miRNA表达谱芯片分析结果的qPCR验证：通过筛选出的“肾虚-非肾虚”组FC较大的差异miRNA,通过PCR方法进一步验证分析结果。结果1.通过对历代中医古籍文献的梳理发现肾虚是消渴发病的重要因素之一。结合表观遗传学方法,能丰富2型糖尿病的肾虚证候微观本质。2.调查2型糖尿病病患793例,有明确家族史的患者169例,占全部21.3%,其发病率无性别差异。有2型糖尿病家族史组的肾虚患者百分比明显高于无2型糖尿病家族史组,两组肾虚患者人数百分比比较有统计学意义,提示“肾虚”可能在2型糖尿病家族聚集中起一定的作用。3.肾虚证、非肾虚证糖尿病及正常对照组三组样本两两比较分别筛选出差miRNA。其中肾虚与非肾虚组差异miRNA13条;肾虚与健康对照差异12条;非肾虚与正常对照差异11条。4.在对“肾虚-非肾虚’miRNA芯片结果中筛选出3条候选差异miRNA:miR-335, miR-377和miR-497,用于qPCR验证。qPCR(?)吉果与芯片结果结果一致,miR-335, miR-377和miR-497在肾虚组中的表达均高于非肾虚组,但组间差异不显著(P>0.05)。结论1.将表观遗传学引入到对中医藏象、证候及中医遗传等很多问题的研究能够有所突破。同时能够更好的阐述“肾为先天之本”的生物学意义。2.通过对2型糖尿病患者家族史的回顾研究发现2型糖尿病存在家族聚集现象,肾虚对2型糖尿病家族聚集趋势起一定的作用。3.2型糖尿病肾虚证在特征性miRNA主要涉及机体免疫及细胞周期等方面有关,提示肾虚在2型糖尿病中可能机理为降低组织细胞功能。4. miRNA表达谱芯片的结果及前期基因芯片结果基本一致,提示2型糖尿病肾虚证的机制涉及多层面的改变,后期有深入研究的必要。
【Abstract】 Objectives:Diabetes has become one of the most important threats to people especially Type2Diabets (T2DM) in our country. The mechanism of T2DM has been proved with complex genetic background and environmental factors. Basd on the traditional Chinese medicine theory "the kidney is the congenital foundation", the study of T2DM mechanism can better reveal the interaction of "gene-environment-disease" and the whole concept of Chinese medicine. In this paper, we tried to explore the epigenetic mechanism in kidney deficiency syndrome of T2DM.1. To discuss the innovation and application value of combination of epigenetics method and Chinese medicine phenomenological research in theory by summary the ancient Chinese medicine book and epigenetic methods.2. To reveal the influence of genetic factors on kidney deficiency through the large scale investigation with kidney deficiency scale.3. To explore the mechanism of kidney deficiency syndrome of diabetes from the molecular level by miRNA microarray.Methods:1. The combination of disease and syndrome:Clinical investigations with kidney deficiency facto scaling on T2DM patients. And the then statistic analyzed the distribution characteristics of kidney deficiency syndrome in T2DM2. miRNA microarray tests:miRNA microarrays screen out potential differentially miRNA for type2diabetes with kidney-deficiency group, non-kidney-deficiency group and normal group. The target gene predicted for the key miRNA.3. Fluorescent quantitation PCR:candidate characristic miRNA in "kidney deficiency group-non kidney deficiency group"were tested with PCR.Results:1. Based on ancient Chinese literature, kidney deficiency is thought to be one of the most important factors in the pathogenesis of diabetes. With epigenetics techology, kidney deficiency syndrome in microscopic nature rich type2diabetes can be enriched.2. In793T2DM patients, there are169cases of patients with family history, accounting for21.3%. There is no dfference in male and femal.The percentage of kidney deficiency patients with a family history was significantly higher than that of no family history (P<0.05). That suggested "deficiency of kidney" may play a role in the familial aggregation of T2DM.3. There are13different miRNA between kidney deficiency group and non-kidney deficiency group; there are12different miRNA between kidney deficiency group and normal controls; there are11different miRNA between non kidney deficiency group and normal controls.4.3candidate different miRNA (miRNA:miR-335.miR-377and miR-497) between kidney deficiency group and non kidney deficiency group were chosen to qPCR verification. The results were consistent with the microarray results. However, the difference between groups was not significant (P>0.05).Conclusions:1. With the help of Epigenetics Chinese medicine research such as Chinese medicine syndromes and genetics may achieve a breakthrough. At the same time the theory "the kidney is the congenital foundation" can be better explained.2. By the investigation of family histories of T2DM patients, the congenital kidney deficiency may play a role in the pathogenesis of T2DM.3. The miRNA basis of kidney deficiency syndrome in T2DM related to the immunity and the aging which may imply kidney deficiency in T2DM possible mechanism to reduce cell function. 4. The miRNA microarray and candidate miRNA qPCR results are basically consistent. It may imply that T2DM deficiency syndrome has certain significance in miRNA level changes which should be necessary to explore deep later.