基于线粒体母系遗传研究肾阳虚体质能量代谢低下的分子机制

From the earlier research supported by NSFC ,we found that there are some similarities as well as some differences in KYD physique transcriptome profile in varies ages(young,mature,old). The slow energy metabolism is the same and the immunodeficiency is different. Because of great individual differences in human, the study couldn't give a definite conclusion. We also found that family clustering with KYD physique is mainly affected by maternalstrain and mutation in mitochondrial DNA origin of replication and promoter region is correlated with KYD. Mitochondria are not only core organelles in energy metabolism, but also have the character of maternal inheritance. So we put forward the hypothesis that 'KYD physique may lead to defects about energy metabolism by mitochondria'. According to select three Chinese pedigrees of KYD physique, we compare patients with KYD physique with partners with normal physique in same pedigree, different ages (young, mature, old) with KYD physique and different ages in different pedigrees in the following index: mitochondrial DNA sequencing, DNA Copy Number, respiratory chain complexes, ATP enzyme content and NA+K+ATP enzyme activity. We study the miRNA expression encoded by core gene with miRNA chip, in order to reveal the correlation mechanism that the occurring of KYD physique interact defects about mitochondrial energy metabolism and the regulatory mechanism that the core gene of KYD physique affect mitochondrial ATP energy metabolism in molecular level. The study can reveal molecular mechanism about congenital physique from maternal inheritance, verify the gene regulatory pattern that kidney Yang wax and wan in different ages(young,mature,old) from similar genetic background, future explain scientific meaning in KYD physique and provide reference to control and keep in good healthy for KYD in clinic.

前自然基金发现肾阳虚证有家族聚集且主要受母系影响，肾阳虚体质转录组谱集中于能量代谢类基因，基因功能富集与线粒体DNA复制启点和重链启动子区的突变有关。因线粒体是能量代谢的核心细胞器，具母性遗传特点，故提出："肾阳虚体质经线粒体途径引起机体能量代谢缺陷"的假说。通过纳入3个肾阳虚体质家系，比较家系内肾阳虚体质与其阴阳平和质配偶间、家系内少/壮/老肾阳虚体质间，不同家系少/壮/老肾阳虚体质间：线粒体的DNA测序、呼吸链酶复合体、ATP酶、膜电位等的异同，结合miRNA芯片检测核基因编码的miRNA表达，从分子水平上揭示肾阳虚体质的发生与线粒体能量代谢缺陷的关联机制，以及伴随着少/壮/老肾阳的盛衰，机体核基因对线粒体ATP能量代谢的基因调控模式。研究从线粒体母系遗传的角度揭示了中医体质先天禀赋遗传的分子机制，阐释了肾阳虚体质病机的科学内涵，为临床肾阳虚体质的防治与养生保生保健提供重要参考。

肾阳虚体质作为偏颇体质之一,与肿瘤、代谢综合征、早衰等病症的关联性开始受到关注。本研究基于前期发现提出：“肾阳虚体质经线粒体途径引起机体能量代谢缺陷”的假说。纳入肾阳虚体质家系比较家系内肾阳虚体质与其阴阳平和质配偶间、家系内少/壮/老肾阳虚体质间、不同家系少/壮/老肾阳虚体质间线粒体能量代谢相关酶学与线粒体全外显子测序的异同。发现：（1）肾阳虚体质组线粒体蛋白含量明显低于平和质组；肾阳虚体质组的线粒体蛋白含量与年龄呈负相关的趋势，平和质组的线粒体蛋白含量与年龄呈正相关的趋势。（2）肾阳虚体质组线粒体ATP酶活性高于平和质组；肾阳虚体质组的ATP酶活性与年龄无明显相关性，平和质组的ATP酶活性与年龄呈负相关的趋势。（3）肾阳虚体质组的线粒体Na+,K+-ATP酶活性与平和质组无明显差异，且与年龄不相关；平和质组的线粒体Na+,K+-ATP酶与年龄呈负相关的趋势。（4）肾阳虚体质组在幼年和老年的膜电位明显低于中年时的膜电位，且与年龄不相关；平和质组的线粒体膜电位与年龄呈正相关的趋势。（5）肾阳虚体质组和平和质组的线粒体呼吸链复合物Ⅰ无明显差异，且与年龄也无明显的相关性。（6）肾阳虚组的线粒体呼吸链复合物Ⅲ样本总活性明显高于平和质组；肾阳虚体质组的样本总活性与年龄呈负相关的趋势，平和质组与年龄无明显相关性。（7）肾阳虚体质组的线粒体呼吸链复合物Ⅴ样本总活性与平和质组无明显差异；肾阳虚体质组和平和质组的线粒体呼吸链复合物Ⅴ的样本总活性与年龄均呈负相关的趋势。（8）线粒体全外显子测序研究筛选出肾阳虚体质线粒体基因179个候选突变，其中23个突变的生物学意义值得关注。在家系研究基础上，建立线粒体DNA文库，纳入18对同为肾阳虚体质的母子/母女，进行线粒体DNA测序，对线粒体母系遗传规律开展进一步验证。研究结果论证了肾阳虚体质与线粒体能量代谢缺陷的密切关联，初步揭示了肾阳虚体质遵循线粒体母系遗传路径对后代体质的影响，初步筛选了肾阳虚体质线粒体突变位点，提示线粒体呼吸链复合物Ⅲ可以作为肾阳虚证临床诊断的候选指标，不仅为肾阳虚体质的诊断以及中医药干预肾阳虚偏颇体质提供了新的靶标，也为减少偏颇体质的优生优育方案提供了方向。