人工光环境对卵巢发育和节律的影响及中药干预作用的研究

This study based on previous research, using artificial light environment to establish rat ovarian cycle rhythm disorder model, compared with natural light environment group and traditional Chinese medicine intervention group. Pathological changes of rat ovary are observed after the effects of light environment by morphology, endocrinology, proteomics, genomics research and so on. Focuse on the impact on the formation of offspring reproductive rhythm also including reversal effect of traditional Chinese medicine intervention. By ovarian clock gene Clock, Bmall and related downstream gene, melatonin receptor gene and its single nucleotide polymorphism analysis watch ovarian development in different light environment, as well as the application of the famous Chinese medicine doctor's effective prescription on rat cycle adjustment function. This study is the first time from the " light environment - ovarian cycle - generation " point of view, focus on changing light environment effects on ovarian development and reproductive ability in rats, and the comprehensive adjustment effect of traditional Chinese medicine intervention, clearly define the intervention effect of traditional Chinese medicine how to correct harmful influence in ovarian development and cycle rhythm established by environment factors.Moreover,the study point out the view that protection of ovarian function in women should be established before puberty ,even in the maternal uterus stag,in order to seek the best time for traditional Chinese medicine intervention before disease happen ,meanwhile provide effective prevention method to resist environmental factors resulting in female reproductive barriers.

本研究在前期研究基础上，采用人工光环境建立大鼠卵巢周期节律紊乱的动物模型，与自然光环境及中药干预组进行比较，通过形态学、内分泌学、蛋白组学、基因组学的研究，观察光环境改变对大鼠卵巢发育和周期节律的影响，并连续追踪对子代生殖功能的干扰，从卵巢钟基因Clock、Bmall和下游基因，褪黑素受体基因及其单核苷酸表达切入，分析光环境改变干扰卵巢发育的途径，并以名老中医经验方逆转大鼠生殖障碍的病理状态，达到调整周期节律的效用。本研究首次从"光环境－卵巢周期节律－子代遗传"角度，重点探讨光环境变化对大鼠卵巢发育及其子代生殖能力的影响，同时阐述中药对卵巢功能的调节作用，明确中药能够纠正环境因素对卵巢发育、周期节律产生的不良影响，提示对女性卵巢功能的保护应当建立在青春期前，甚至在母体宫内阶段，为中医药在生殖内分泌领域"治未病"寻求最佳干预时机，是抵御环境因素造成女性生殖障碍性疾病的有效方法。

目的：探讨光污染对大鼠卵巢发育和节律的影响及滋阴补阳方序贯的治疗作用。方法：通过给予大鼠不同的光照强度，探索生殖节律紊乱模型造模的最佳方法；给予造模大鼠滋阴补阳序贯中药治疗，观察体重、动情周期、性激素及褪黑素的变化；对比模型组、正常组、中药组大鼠性腺、松果体形态学；卵巢褪黑素受体及生物钟基因的表达；对三组大鼠卵巢进行miRNA高通量测序，对有显著差异表达的miRNA进行荧光定量PCR实验；以荧光素酶报告基因，验证miRNA-421-5p与MAPK7的作用关系；同时以慢病毒载体介导shRNA干扰miRNA-421-5p探讨MAPK信号通路上蛋白的表达。结果：以300±20lux光强持续照射50天，获得大鼠生殖节律紊乱模型，应用滋阴补阳方序贯疗法恢复了卵巢、子宫、松果体结构、提高大鼠体重、卵巢湿重和近日节律。模型组的miRNA-421-5p表达与对照组相比呈下调状态，中药组miRNA-421-5p较模型组表达上调，验证了miRNA-421-5p与MAPK7的关系，当miRNA-421-5p过表达时，MAPK7以及下游通路上的c-fos、creb、p-creb、c-myc表达下调；当miRNA-421-5p基因沉默时，MAPK7以及下游通路上的c-fos、creb、p-creb、c-myc表达上调。结论：滋阴补阳方序贯疗法可在一定程度上改善生殖内分泌功能、提高卵巢重量和恢复近日节律；其效应可能是通过调控miRNA-421-5p靶向作用于MAPK的信号通路，改善卵巢功能、提高卵巢重量以及恢复卵巢正常的结构。