Research on the Isolation of Mouse Leydig Cells Using Differential Digestion with a Low Concentration of Collagenase
Research on the Isolation of Mouse Leydig Cells Using Differential Digestion with a Low Concentration of Collagenase
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DOI：
10.1262/jrd.10-123n
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发表时间：
2011-06-01
影响因子：
1.8
通讯作者：
Liu, Guohua
中科院分区：
文献类型：
--
作者：
Sun, Jie;Zhong, Liang;Liu, Guohua
本研究旨在建立一种分离纯化小鼠睾丸间质细胞的新方法。收集青春期后小鼠睾丸，用低浓度胶原酶NB 4消化15 min 2次。将获得的细胞在具有10%FBS的低葡萄糖DMEM中培养。免疫荧光法检测睾丸间质细胞生物标志物3 β-羟基类固醇脱氢酶、胆固醇侧链裂解酶（CYP 11 A1）和17 α-羟化酶/17，20-裂解酶（CYP 17 A1）的表达。结果表明，分离的睾丸间质细胞纯度为69.6 ± 4.16%。在原代培养7天后，其增加至90%。在原代培养中检测到睾酮合成酶谱。总之，应用低浓度的胶原酶进行差异消化可以分离出大量的活Leydig细胞。
The aim of this study was to establish a novel method for isolating and purifying Leydig cells from mice testes. Testes of postpuberal mice were harvested and digested in a low concentration of collagenase NB4 for 15 min 2 times. Cells obtained were cultured in low glucose DMEM with 10% FBS. Immunofluorescence was used to detect the expression of Leydig cell biomarkers including 3 beta-hydroxysteroid dehydrogenase, cholesterol side-chain cleaving enzyme (CYP11A1) and 17 alpha-hydroxylase/17,20-lyase (CYP17A1). It was found that the purity of the isolated Leydig cells was 69.6 +/- 4.16%. After 7 days in primary culture, it increased to 90%. The testosterone synthase spectrum could be detected at the primary culture. In conclusion, the application of a low concentration of collagenase for differential digestion allows isolation of large quantities of viable Leydig cells.