多囊卵巢综合征患者卵泡液外泌体的提取鉴定及其miRNAs的提取和检测

发布时间：2018-08-19 20:14

【摘要】：目的·从多囊卵巢综合征患者卵泡液中分离提取外泌体并进行鉴定,明确卵泡液中外泌体的存在;从外泌体中分离提取miRNAs并检测其表达量。方法·利用膜亲和法提取卵泡液中外泌体,透射电镜观察大小和形态;利用流式细胞仪检测外泌体蛋白标志物CD63和CD81的表达;提取纯化外泌体miRNAs,利用Taq Man探针real-time PCR检测miR-125b、miR-19b和miR-222的表达。结果·透射电镜下观察到卵泡液外泌体呈圆形或椭圆形,直径30~100 nm,有完整的膜结构,内含低密度物质;流式细胞仪检测外泌体CD63和CD81呈阳性表达。Real-time PCR检测到外泌体中miR-125b、miR-19b和miR-222的表达。结论·多囊卵巢综合征患者卵泡液中可以收集到外泌体,可以通过电子显微镜和流式细胞仪对卵泡液中的外泌体进行鉴定。卵泡液外泌体中含有miR-125b、miR-19b和miR-222。
[Abstract]:Objective to isolate and identify the exocrine bodies from the follicular fluid of patients with polycystic ovary syndrome (PCOS), to identify the presence of the exocrine bodies in the follicular fluid, and to isolate and detect the expression of miRNAs from the exocrine bodies of the patients with polycystic ovary syndrome (PCOS). Methods Follicular fluid was extracted by membrane affinity method. The size and morphology of follicular fluid were observed by transmission electron microscope (TEM), and the expression of CD63 and CD81 were detected by flow cytometry. The exocrine miRNAswere extracted and purified, and the expression of miR-125b miR-19b and miR-222 was detected by Taq Man probe real-time PCR. Results under the transmission electron microscope, the exocrine body of follicular fluid was round or oval, with a diameter of 30 ~ 100 nm, a complete membrane structure and a low density substance. The positive expression of CD63 and CD81 in exocrine was detected by flow cytometry. Real-time PCR was used to detect the expression of miR-125bnmiR-19b and miR-222 in exocrine. Conclusion the exocrine bodies can be collected in the follicular fluid of patients with polycystic ovary syndrome, and the exocrine bodies in the follicular fluid can be identified by electron microscope and flow cytometry. The follicular exocrine contains miR-125b miR-19b and miR-222.
【作者单位】：上海交通大学医学院附属国际和平妇幼保健院胚胎源性疾病研究所;浙江大学医学院附属妇产科医院生殖遗传教育部重点实验室;
【基金】：国家科技支撑计划(2012BAI32B00) 国家自然科学基金(81450038)~~
【分类号】：R711.75

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