【作者】 赵娟；

【导师】 谈勇；

【作者基本信息】 南京中医药大学 ， 中医妇科学， 2013， 博士

【摘要】 目的：通过研究滋阴补阳中药序贯对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞(GC)分泌功能和细胞因子基因表达的调控,以及滋阴补阳中药序贯对PCOS不孕患者促排卵周期、体外受精-胚胎移植(IVF-ET)中血清、卵泡液中干细胞因子(SCF)水平的影响及其与优势卵泡、卵细胞质量的关系,探讨滋阴补阳中药序贯对PCOS干预的作用机理。方法：1.实验研究：将造模成功的PCOS大鼠,按实验要求分离与培养卵巢颗粒细胞,并制备空白血清、滋阴方、补阳方含药血清,观察滋阴方、补阳方对PCOS大鼠卵巢颗粒细胞分泌E2、P功能的影响,以及对干细胞因子(SCF)、雄激素受体(AR)、类固醇合成急性调控蛋白(StAR)基因表达调控的影响。2.临床研究：选取PCOS患者60例,其中经典PCOS30例,无高雄PCOS30例；并选取非PCOS患者30例作为正常组对照,分析基础状态下SCF与PCOS发病机制的相关性。另选取62例行促排卵治疗的PCOS患者随机分为2组,治疗组(滋阴补阳中药序贯／克罗米芬(CC)／果纳芬(r-FSH),30个周期)和对照组(CC/r-FSH,32个周期)。观察两组患者HCG注射天数、子宫内膜厚度、优势卵泡个数、HCG日血清E2、LH、SCF水平、比较两组在排卵率、妊娠率、卵巢过度刺激综合征(OHSS)以及卵泡未破裂黄素化综合征(LUFS)等方面的效果。40例将行IVF-ET的PCOS患者随机分为2组,治疗组(滋阴补阳中药序贯联合西药组)和对照组(西药组),每组各20例。观察两组患者HCG日血清E2、LH、P水平、取卵日卵泡液中SCF水平以及取卵个数、受精率、卵裂率、优质胚胎率、妊娠率。结果：1.造模后,PCOS大鼠卵巢大体解剖以及HE染色后卵巢光镜均有明显不同。2.卵泡期中,滋阴方10%、20%组均可有效提高PCOS大鼠颗粒细胞上清液中的E2、SCF含量；黄体期中,补阳方10%组刺激黄素化颗粒细胞分泌P的作用最强。当补阳方浓度继续增大,其促进P分泌的作用并没有增强。3.模型组PCOS大鼠颗粒细胞分泌E2、P水平显著降低。10%、20%滋阴方、20%补阳方均可有效促进PCOS大鼠卵巢颗粒细胞分泌E2、P,滋阴方的浓度与颗粒细胞分泌E2、P成一定的量效关系。4.模型组大鼠卵巢GC中ARmRNA表达显著升高,SCF、StARmRNA表达显著降低。10%、20%滋阴方组、20%补阳方组可有效下调ARmRNA表达,上调SCF、StARmRNA表达。5.无高雄PCOS AFC明显多于经典PCOS组、正常组。无高雄PCOS组与正常组T水平显著低于经典PCOS组。无高雄PCOS组血清SCF水平明显升高。无高雄PCOS组SCF水平与AFC呈正相关。6.PCOS患者促排卵治疗后,试验组治愈率36.7%,总有效率86.7%；对照组治愈率12.5%,总有效率87.5%,两组治愈率有显著差异。试验组HCG日子宫内膜厚度、HCG日≥14mm卵泡数、HCG日≥18mm卵泡数均有显著差异。试验组HCG日血清E2、SCF明显高于对照组。7. IVF-ET周期治疗组取卵日卵泡液中SCF水平、受精率、优质胚胎率明显高于对照组。妊娠组取卵日卵泡液SCF水平显著高于未妊娠组。结论：1.SCF与PCOS卵泡发育障碍的发病机制相关。2.滋阴、补阳方可明显改善PCOS大鼠卵巢颗粒细胞分泌功能,下调AR基因表达,上调SCF、StAR基因表达。3.滋阴补阳中药序贯联合西药促排卵可以增加子宫内膜厚度及HCG日E2、SCF水平,提高妊娠率。4.对PCOS患者IVF-ET中进行滋阴补阳中药序贯干预,可改善卵巢内微环境,提高卵泡液内SCF水平,从而提高受精率、优质胚胎率、妊娠率。更多还原

【Abstract】 Objective:Through sequential therapy of nourishing Yin and supplementing Yang of traditional Chinese medicine intervention to study the mechanism of the therapy controlling secretion regulation function and expression of cytokine genes in ovarian granulosa cells in PCOS rats.Furthermore, monitor stem cell factor(SCF) level in serum,follicular fluid by ovulation induction cycle or in vitro fertilization and embryo transfer (IVF-ET) cycle on infertile patients with PCOS,besides its relationship with the influence of dominant follicle and oocyte quality.Methods:1. Experiment Study:According to the experimental requirements establish PCOS rats model,then, isolate and culture ovarian granulosa cells in different periods.Prepare three different medicated serum namely blank serum, nourishing Yin serum, supplementing Yang serum, to observe their effects on PCOS rats’ovarian granulosa cell secretion function of E2, P,and the influence of stem cell factor, androgen receptor, steroidogenic acute regulatory protein gene expression.2. Clinical research:30cases of classic PCOS,30cases PCOS without hyperandrogenism,30patients without PCOS were selected to analysis correlation of SCF and PCOS pathogenesis in basic state. Another62cases PCOS patients are randomly divided into the treatment group (sequential therapy of nourishing Yin and supplementing Yang of traditional Chinese medicine/clomiphene citrate/Gonal-F,30cycles) and control group (clomiphene citrate/,Gonal-F,32cycles).Two groups were observed with treatment days, endometrial thickness, the dominant follicles number,serum E2, LH, SCF levels in the HCG injection day and compared ovulation rate, pregnancy rate,ovarian hyperstimulation syndrome (OHSS) and luteinized unruptured follicle syndrome (LUFS) etcetera.40cases of PCOS patients of IVF-ET were randomly divided into into2groups, experimental group (sequential therapy of nourishing Yin and supplementing Yang of traditional Chinese medicine with western medicine group) and control group (Western medicine group), each group of20cases. Observation serum E2, LH, P levels in HCG injection day,SCF levels in follicular fluid and the difference of retrieved oocyte, fertilization rate, cleavage rate, high quality embryo rate and pregnancy rate.Results:1. PCOS rat ovarian anatomy and HE staining light microscope ovarian are obviously different.2. In follicular phase, nourishing Yin Decoction10%,20%groups can improve PCOS rats’E2, SCF content in granulosa cell,similarly in luteal phase, supplementing Yang Decoction20%group displayed the strongest function of stimulating granulosa cells to secret P,but no enhancement when concentration increasing. No statistical significance among the groups of SCF level.3. There were significantly reduced the granulosa cells secretion of E2and P in model group.Both nourishing Yin Decoction10%,20%groups and supplementing Yang Decoction20%group can promote PCOS rats’ ovarian granulosa cells to secret E2, P.And that nourishing Yin Decoction concentration has a certain dose-effect relationship with granulosa cell secretion level of E2, P.4. Expression of AR mRNA in model group was significantly increased, but SCF, StAR mRNA expression decreased obviously.while,nourishing Yin Decoction10%,20%groups and supplementing Yang Decoction20%group can effectively reduce the granulosa cell AR mRNA expression, increase SCF, StAR mRNA expression.5. The without hyperandrogenism PCOS group AFC was significantly more than the classical PCOS group, normal group,and had a significantly elevated SCF levels,show a positive correlation with AFC. But,its T level,besides the normal group,was significantly lower than the classical PCOS group.6. The treatment group’s cure rate was36.7%and total efficiency of86.7%,meanwhile,control group, the cure rate was12.5%, the total efficiency of87.5%, two group had significant difference in the cure rate, endometrial thickness,above14mm and18mm follicles number in HCG injection day. Treatment group’s serum E2, SCF level in HCG injection day were significantly higher than those in control group. The test group was higher than that of the control group pregnancy.7. In IVF-ET cycles, the follicular fluid levels of SCF in retrieved oocyte day, fertilization rate, high quality embryo rate was significantly higher in treatment group han that of control group. In either group,pregnant patients’follicular fluid SCF levels were significantly higher than those not pregnant in retrieved oocyte day.Conclusion1. SCF relates to the pathogenesis of PCOS follicles developmental disorders.2. Sequential therapy of nourishing Yin and supplementing Yang of traditional Chinese medicine intervention can significantly improve the PCOS rats’ ovarian granulosa cells secretion function, down-regulate AR gene expression, up-regulate SCF, StAR gene expression.3. Sequential therapy of nourishing Yin and supplementing Yang of traditional Chinese medicine with western medicine to induce ovulation can increase the endometrial thickness, serum E2, SCF levels in HCG injection day, improve the pregnancy rate.4. IVF-ET patients with PCOS,intervent by sequential therapy of nourishing Yin and supplementing Yang of traditional Chinese medicine, can improve the ovarian microenvironment, improve the SCF level in follicular fluid, so as to improve the fertilization rate, high quality embryo rate, pregnancy rate.更多还原