Isolation of granulosa cells from follicular fluid; applications in biomedical and molecular biology experiments.
Isolation of granulosa cells from follicular fluid; applications in biomedical and molecular biology experiments.
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DOI：
10.4103/2277-9175.170675
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发表时间：
2015
影响因子：
1
通讯作者：
Nouri M
中科院分区：
文献类型：
--
作者：
Aghadavod E;Zarghami N;Farzadi L;Zare M;Barzegari A;Movassaghpour AA;Nouri M
近年来，人们对含颗粒细胞卵母细胞质量低下的分子机制进行了大量的研究。GC是卵泡液中发现的主要细胞类型之一，从卵泡液中纯化这些细胞对于进一步研究非常重要。虽然有不同的纯化技术，但分离高纯度和损伤最小的细胞的方法是必要的。本文提出了一种高纯度、高产量气相色谱分离的新方法。收集接受体外受精的患者的卵泡液，采用Ficoll、Percoll和红细胞裂解液（RLB）法提取卵泡液中的GC。用流式细胞仪检测提取的GCs纯度，用差示干涉显微镜观察GCs的形态学特征。通过NanoDrop 1000、预限制性片段长度多态性和电泳技术检查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，影响了GC提取的质量和数量。结果表明，不同的纯化方法对细胞的提取量和质量均有影响。与Ficoll法和Percoll法相比，RLB法是一种简便的GC提取方法。
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.