Aghadavod Esmat, Zarghami Nosratollah, Farzadi Laya, Zare Mina, Barzegari Abolfazl, Movassaghpour Ali Akbar, Nouri Mohammad
Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan, Iran; Women's Reproductive Health Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
Department of Biochemistry and Clinical Laboratories, Tabriz University of Medical Sciences, Tabriz, Iran.
Adv Biomed Res. 2015 Nov 30;4:250. doi: 10.4103/2277-9175.170675. eCollection 2015.
Recently, a lot of research has been conducted to investigate the molecular mechanisms of the low quality of oocytes with granulosa cells (GCs). GCs are one of the major cell types found in follicular fluid and purification of these cells from the follicular fluid is very important for further studies. Although, there are different techniques of purification, a method for separation of highly-pure and minimally-damaged cells is necessary. In this paper, we presented a novel method for high purification of GCs with a large quantity and high purity.
Follicular fluid was collected from patients who referred for in vitro fertilization and GCs in follicular fluid were extracted by Ficoll, Percoll and Red blood cell lysing buffer (RLB) methods. Then purity of extracted GCs was assessed by flow cytometry and morphological properties of GCs were observed by differential interference contrast microscopy. The purity of deoxyribonucleic acid and ribonucleic acid extracts was examined by NanoDrop 1000, pre-restriction fragment length polymorphism and electrophoresis techniques. Quality and quantity of extracting GCs were affected during the cell separation procedures.
Our results showed that each of purification method can affect quality and quantity of extracted cells.
RLB method for extraction of GCs was shown to be a convenient procedure in comparison with Ficoll and Percoll methods.
最近，已经开展了大量研究来探究颗粒细胞（GCs）导致卵母细胞质量低下的分子机制。颗粒细胞是卵泡液中主要的细胞类型之一，从卵泡液中纯化这些细胞对于进一步研究非常重要。尽管有不同的纯化技术，但需要一种能够分离出高纯度且损伤最小的细胞的方法。在本文中，我们提出了一种高纯度大量纯化颗粒细胞的新方法。
从接受体外受精的患者中收集卵泡液，采用Ficoll、Percoll和红细胞裂解缓冲液（RLB）方法提取卵泡液中的颗粒细胞。然后通过流式细胞术评估提取的颗粒细胞的纯度，并通过微分干涉相差显微镜观察颗粒细胞的形态学特性。通过NanoDrop 1000、预限制性片段长度多态性和电泳技术检测脱氧核糖核酸和核糖核酸提取物的纯度。在细胞分离过程中，提取颗粒细胞的质量和数量受到影响。
我们的结果表明，每种纯化方法都会影响提取细胞的质量和数量。
与Ficoll和Percoll方法相比，RLB法提取颗粒细胞是一种简便的方法。
