基于线粒体能量代谢路径探讨«内经»“五七”之后生殖衰老“肾虚”本质

With the promotion of the universal two-child policy, the fertility problem of older women has attracted more and more attention.The theory of Traditional Chinese Medicine in Huangdi Neijing has an exposition of the relationship between age and female reproductive function.It discusses the physiological changes of women at all ages, and emphasizes that the maintenance of female reproductive function is dominated by kidney qi. It is a basic understanding of Traditional Chinese Medicine for female reproductive function.The Seven-Seven theory contains a profound and scientific connotation, but is short of essential interpretation.The function of mitochondrial energy metabolism has become an objective evaluation index for the quality of oocyte, and it is of great significance in assessing female reproductive function.This subject is aimed at excavating the scientific connotation of the Seven-Secen theory.We intend to use the Time-lapse Imaging(TLI) to make a morphological and energetical analysis,using the 'Three-Seven' to 'Six-Seven' ages IVF-ET patients' oocytes and early embryos.We test Mfn2 in the granulosa cells of follicular fluid and use NICS technique to test mtDNA copy number of embryo culture supernatants.At the same time, we use classical tonifying kidney prescription to test pattern of syndrome and analyze the oocyte, embryo development and pregnancy outcome by using mitochondrial energy metabolism pathway.This subject provides theoretical and experimental basis for us to forecast and diagnose the kidney deficiency's essence and reproductive function decline after the age of 'Five-Seven'.It provides a new idea for clinical Traditional Chinese Medicine to improve the outcome of the elderly in vitro fertilization and embryo transfer.It also contributes to the solution of the fertility problem of the elderly infertile couples.

随着二胎政策的全面放开，高龄女性的生育问题越来越受关注。中医理论早在《内经》中就对年龄与女性生殖功能关系进行阐述，论述女性各年龄阶段的生理变化，强调女性生殖机能的维持以肾气为主导，是中医学对女性生殖功能的基本认识。但对“七七”理论所蕴含的丰富科学道理缺乏深入本质的阐释。线粒体能量代谢功能已成为反应卵母细胞质量的客观评价指标，对评估女性生殖功能意义重大。本课题以挖掘“七七”理论的科学内涵为目标，通过对“三七”到“六七”年龄段IVF患者卵母细胞及早期胚胎实时监控进行形态动能学分析，卵泡液颗粒细胞Mfn2的检测及运用NICS技术对胚胎培养液mtDNA拷贝数的检测，并用补肾经方干预治疗以方测证，从线粒体能量代谢路径对卵母细胞、胚胎发育及妊娠结局进行分析。为科学预测和诊断“五七”之后生殖衰老“肾虚”本质研究提供理论和实验依据，为临床中医药改善高龄体外受精-胚胎移植治疗结局提供新思路，为解决高龄不孕夫妇的生育问题作出贡献。

本研究从《内经》“七七”理论出发，从线粒体能量代谢途径研究“五七”后高龄女性生殖功能衰退的机理，方证结合，研究补肾中药对高龄女性线粒体相关的关键靶点的作用，并与时差成像技术相结合，观察补肾中药对高龄女性体外受精-胚胎移植（IVF-ET）患者胚胎发育时间参数和卵裂模式的调控作用，并构建了补肾中药治疗高龄女性不孕的药物-疾病-靶点调控网络。.结果显示随着女性年龄的增长，卵母细胞数、2PN数、胚胎数、子宫内膜厚度、临床妊娠率、内膜厚度、Mfn1（线粒体融合蛋白1）和Mfn2（线粒体融合蛋白2）及mRNA表达均下降，Drp1蛋白水平及基础卵泡刺激素（FSH）水平升高，证实了“七七 ”理论的科学依据理。应用时差成像技术观察补肾中药对高龄体外受精-胚胎移植（IVF-ET）患者胚胎发育时间参数的调控作用，在t5时间段中，正常对照组及中药治疗组均明显快于安慰剂组，且中药治疗组肾虚症状明显改善，中药治疗组2PN受精率高于安慰剂组。从网络药理学的层面观察补肾中药经验方二至天癸方对于高龄女性不孕症的干预机制，发现其主要活性成分与 Mfn-1、Mfn-2 较好结合，验证了补肾中药基于线粒体能量代谢途径对高龄女性不孕症的治疗作用，为高龄女性不孕症的中医药治疗提供系统生物学信息和基础。.本研究阐述了“五七”后女性生殖衰老的生物学基础，从线粒体能量代谢层面揭示了高龄不孕患者的证候要素和补肾中药的作用靶点，创新性的将非侵入性胚胎检测技术与中医药结合，丰富和发展了《内经》“七七”理论，为高龄女性的生育困境提供了中医药策略。