College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
Tissue Cell. 2013 Aug;45(4):269-74. doi: 10.1016/j.tice.2013.03.004. Epub 2013 Apr 29.
As an important cell model, luteal cells are used to study the reproductive cycle and pregnancy maintenance, but there has not yet had a simple and economical method in purifing goat luteal cells. In order to find a good method to isolate and purify the luteal cells from the Guan Zhong dairy goat corpus luteua, we compared the purification efficiency of Percoll density gradient centrifugation method with that of the differential detachment method using trypsin. After using these two methods for isolation, the purified cells were identified by staining for 3β-hydroxy steroid dehydrogenase activity. Cell diameter measurements and cell counting were used to categorize isolated cells from both methods. Cell proliferation activity of purified cells from both methods were studied by Cell Counting Kit-8 for 8 days. The results showed that, after Percoll discontinuous density gradient centrifugation, the purity of luteal cells was 98.2±1.2% in Percoll density layer of 30-40%. In comparison, the purity of luteal cells isolated in differential detachment by trypsin was 74.3±1.8%. Luteal cells purified from both methods stained positive for 3β-hydroxy steroid dehydrogenase activity, and cells purified by Percoll centrifugation showed a more rapid cell proliferation rate than cells purified by trypsin. In conclusion, this study has demonstrated that Percoll density gradient centrifugation was superior to the method of differential detachment in cell purification efficiency and in maintenance of cell proliferation activity.
作为一种重要的细胞模型，黄体细胞被用于研究生殖周期和妊娠维持，但目前还没有一种简单、经济的方法来纯化山羊黄体细胞。为了找到一种从关中奶山羊黄体中分离和纯化黄体细胞的好方法，我们比较了聚蔗糖（Percoll）密度梯度离心法和用胰蛋白酶进行差异分离法的纯化效率。使用这两种方法进行分离后，通过 3β-羟类固醇脱氢酶活性染色来鉴定纯化的细胞。通过细胞直径测量和细胞计数来对两种方法分离的细胞进行分类。使用细胞计数试剂盒-8 对纯化细胞的增殖活性进行了 8 天的研究。结果表明，在聚蔗糖不连续密度梯度离心后，聚蔗糖 30-40%密度层中的黄体细胞纯度为 98.2±1.2%。相比之下，用胰蛋白酶进行差异分离的黄体细胞纯度为 74.3±1.8%。两种方法纯化的黄体细胞 3β-羟类固醇脱氢酶活性染色均为阳性，用 Percoll 离心法纯化的细胞增殖速度比用胰蛋白酶纯化的细胞更快。综上所述，本研究表明，与差异分离法相比，聚蔗糖密度梯度离心法在细胞纯化效率和维持细胞增殖活性方面更具优势。
