【摘要】 目的 评价卵泡液（follicular fluid,FF）外泌体miRNA对颗粒细胞（granulosa cells,GCs）糖酵解途径介导的多囊卵巢综合征（polycystic ovary syndrome,PCOS）患者卵泡发育的影响，并探讨其作用机制。方法 招募3名PCOS不孕患者和3名非PCOS不孕患者，测定2组患者在促排卵前的基线激素水平，通过短效长方案促排卵并穿刺取双侧FF，超速离心法收集外泌体，采用透射电镜进行鉴定。Trizol法提取外泌体总RNA，构建文库，进行miRNA测序及质控处理后，与参考基因组GRCh38进行比对。利用聚类Profiler R包进行差异表达基因（differentially expressed genes,DEGs）的GO注释分析和KEGG通路分析。应用Omnipath数据库对miRNA进行互作分析。随机抽取16个miRNA，通过qPCR检测，验证miRNA测序结果的准确性。结果 与非PCOS组比较，PCOS组的黄体生成素（luteinizing hormone,LH）、抗缪勒管激素（anti-Mullerian hormone,AMH）、睾酮（testosterone）、窦卵泡数显著高于非PCOS组（t=2.479～9.163,P均<0.05）。2组FF外泌体边缘均清晰，中央淡染的杯口状囊泡，直径为100～150 nm，结构完整，外泌体浓度约为8×1010particles/mL。miRNA测序共检测到928个miRNA，与非PCOS组比较，POCS组外泌体中共筛选出59个差异miRNA(DEmiRNA)，其中31个上调，28个下调，且qPCR法检测的基因表达差异趋势与miRNA测序结果高度相似。在PCOS患者FF外泌体中，通过miRNA调控mRNA，可显著改变GCs糖酵解效率和细胞凋亡状态。PKM、PFKL、HK2是miRNA调节GCs糖酵解的关键靶基因，SLC2A1是miRNA调节GCs凋亡的关键靶基因。结论 PCOS患者FF外泌体miRNA可使GCs糖酵解减弱，并加速其凋亡，从而减少ATP及乳酸的产生，导致卵泡发育障碍。更多还原

【Abstract】 Objective To evaluate the effect of follicular fluid(FF)exosomal miRNAs on follicular dysplasia in patients with polycystic ovary syndrome(PCOS)mediated by glycolysis pathway of granulosa cells(GCs),and to explore the mechanism. Methods Three PCOS infertile patients and three non-PCOS infertile patients were recruited. The baseline hormone levels of the two groups were measured before ovulation induction. The bilateral FF was obtained by puncture after short-acting and long-term ovulation induction,and the exosomes were collected by ultracentrifugation and identified by transmission electron microscopy. The total exosomal RNA was extracted by Trizol method to construct the library,which was compared to the reference genome GRCh38 for statistical analysis after miRNA sequencing and quality control processing. Clustering Profiler R package was used to implement GO annotation analysis and KEGG pathway analysis of the differentially expressed genes(DEGs),and Omnipath software for miRNAs interaction analysis. A total of 16 miRNA were randomly selected and detected by qPCR to verify the accuracy of the miRNA sequencing results. Results Compared with the non-PCOS group,luteinizing hormone(LH),anti-Muerian hormone(AMH),testosterone and antral follicle counts in PCOS group increased significantly(t = 2. 479 ～ 9. 163,each P < 0. 05). The exosomes of FF in both groups showed the cup-shaped vesicles with clear edge and light staining in the center,with the diameters of 100 — 150 nm and intact structure,and the concentration was about 8 × 1010particles/mL. A total of 928 miRNAs were detected by miRNA sequencing. Compared with the non-PCOS group,59 differentially expressed miRNA(DEmiRNA)were screened out in exosomes of POCS group,of which 31 were up-regulated and 28 were down-regulated. The differential trend of gene expression detected by qPCR was highly similar to that of miRNA sequencing. In FF exosomes of PCOS patients,the glycolysis efficiency and apoptosis of GCs were significantly changed by miRNA regulating mRNA. PKM,PFKL and HK2 were the key target genes for miRNA to regulate GCs glycolysis,and SLC2A1 was the key target gene for miRNA to regulate GCs apoptosis. Conclusion The miRNAs in FF exosomes of PCOS patients can weaken the glycolysis of GCs while accelerate the apoptosis,thus reducing the production of ATP and lactic acid,resulting in follicular dysplasia.更多还原