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Abstract：
The aim of the current study was to develop an accurate and reproducible method for isolation of granulosa cells (GCs) in patients with different ovarian reserves. The cells of healthy individuals and patients with polycystic ovary syndrome (PCOS) were isolated using a modified two-step Percoll density gradient centrifugation. The cells of patients with poor ovarian response (POR) were isolated using a one-step method suitable for samples containing few cells. Cells extracted using these purification techniques were compared regarding cell yield, viability and purity using immunocytochemistry, flow cytometry, Cell Counting Kit-8, western blotting and RNA integrity analysis. The purity and activity of the cells in the POR group were comparable with those in the healthy and PCOS groups and no statistically significant differences were identified. Furthermore, isolated cells analyzed for RNA integrity indicated good quality RNA and presented an RNA integrity number of 8-10, demonstrating that the technique enabled the isolation of GCs from different types of patients. Thus, a reliable and reproducible technique for the isolation of pure GCs with high yield is described in the present study. This protocol provides an efficient method targeted to patients with different ovarian reserve functions that enables the preparation of GCs for evaluating their molecular functions.
摘要：
本研究的目的是开发一种准确且可重复的方法，用于分离具有不同卵巢储备的患者的颗粒细胞（GC）。使用改良的两步Percoll密度梯度离心法分离健康个体和多囊卵巢综合征（PCOS）患者的细胞。使用适用于含有少量细胞的样品的一步法分离卵巢反应不良（POR）患者的细胞。比较使用这些纯化技术提取的细胞的细胞产量，使用免疫细胞化学的活力和纯度，流式细胞术,细胞计数试剂盒-8，蛋白质印迹和RNA完整性分析。POR组的细胞纯度和活性与健康组和PCOS组相当，没有统计学上的显著差异。此外，分析RNA完整性的分离细胞显示出高质量的RNA，并呈现8-10的RNA完整性数，表明该技术能够从不同类型的患者中分离GC。因此，本研究描述了一种可靠且可重复的高收率分离纯GC的技术。该方案提供了一种针对具有不同卵巢储备功能的患者的有效方法，该方法能够制备GC以评估其分子功能。
