Isolation and identification of ovarian theca-interstitial cells and granulose cells of immature female mice.
Isolation and identification of ovarian theca-interstitial cells and granulose cells of immature female mice.
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未成熟雌性小鼠卵巢卵泡膜间质细胞和颗粒细胞的分离和鉴定。
DOI：
10.1002/cbin.10426
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Luo Aiyue
中科院分区：
文献类型：
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作者：
Tian Yong;Shen Wei;Lai Zhiwen;Shi Liangyan;Yang Shuhong;Ding Ting;Wang Shixuan;Luo Aiyue
卵泡间质细胞（tic）和颗粒细胞（GCs）是卵泡的重要组成部分，支持卵泡发育和激素分泌，被认为是基础研究的重要细胞模型。然而，目前还没有方法可以同时从未成熟小鼠的单个卵巢中分离出tic和GCs。在这里，我们试图开发这样一种方案，使用机械解剖结合短暂的胶原酶- dna酶消化。通过形态学特征和分子标记鉴定tic和gc。在分离的tic中，胆固醇侧链切割细胞色素P450 （P450scc）大量表达，而抗苗勒管激素（AMH）仅在非常低的水平表达。这种表达谱在GCs中是相反的。此外，tic分泌大量睾酮(T)和少量雌二醇（E2），而GCs则相反。随着促黄体生成素（LH）浓度的增加，TIC培养基中T浓度逐渐升高。在GCs中，e2的分泌随着促卵泡激素（FSH）浓度的增加而增加。因此，机械解剖结合胶原酶- dna酶消化是一种简单、有效和可重复的方法，可从一个卵巢中获得大量高纯度和激素刺激的tic和GCs。
Theca‐interstitial cells (TICs) and granulosa cells (GCs) are important components of follicles that support follicle development and hormone secretion, and are considered to be important cell models for basic research. However, no method currently exists for simultaneously isolating TICs and GCs from a single ovary of the immature mouse. Here, we sought to develop such a protocol using mechanical dissection combined with brief collagenase‐DNase digestion. Morphological characteristics and molecular markers were detected to identify TICs and GCs. In isolated TICs, cholesterol side chain cleavage cytochrome P450 (P450scc) was expressed abundantly, but anti‐Mullerian hormone (AMH) was expressed only at very low levels. This expression profile was reversed in GCs. In addition, TICs secreted large amounts of testosterone (T) and minimal amounts of estradiol (E2), while the converse was found in GCs. T concentrations rose gradually in TIC culture media as the concentration of added luteinizing hormone (LH) was increased. In GCs, E2secretion increased as the follicle‐stimulating hormone (FSH) concentration increased. Thus, mechanical dissection combined with collagenase‐DNase digestion is a simple, effective and reproducible method for obtaining large numbers of highly purified and hormonally stimulated TICs and GCs from one ovary.