小鼠原代胸腺上皮细胞分离方法的比较与优化

Comparison and optimization of isolation methods of mouse primary thymus epithelial cells

目的比较几种小鼠原代胸腺上皮细胞（TEC）分离方法的效果，筛选简单、高效、实用的分离方法。方法将小鼠胸腺组织根据所用的消化分离方法分为以下几组：胶原酶Ⅰ组 （Collagenase Ⅰ, Col IE组, 1 mg/ml）、DispaseⅡ组 （DisⅡ组, 3 mg/ml）、Col IE＋低浓度DisⅡ组 （Col IE/Dis Ⅱ-L组, 0.75 mg/ml）、Col IE＋中浓度DisⅡ组 （Col IE/Dis Ⅱ-M组, 1.5 mg/ml）、Col IE＋高浓度DisⅡ组（Col IE/DisⅡ-H组, 3 mg/ml）。优选出最适消化方法后，将所得单细胞悬液分成Percoll实验组以及Percoll对照组。采用血细胞计数板进行总细胞数计数，台盼蓝染色法观察细胞的活性；使用流式细胞术检测胸腺基质细胞（TSC）以及TEC的比例。结果与单独使用DisⅡ与Col IE相比，Col IE与不同浓度Dis Ⅱ联合使用均能加快胸腺组织消化（P〈0.01），获取单个胸腺的总细胞数较多，尤以中、高浓度DisⅡ与Col IE联合消化效果更佳（P〈0.05或P〈0.01）。与单独使用酶消化组相比，Col IE与中、高浓度DisI联合消化均可不同程度地增加获取TSC和TEC的比例（P〈0.01）。与Percoll对照组相比，Percoll密度梯度离心进一步提高获取TSC和TEC的比例（P〈0.05）。此外，各种消化分离方法均可获得高活细胞率的细胞。结论Col IE与DisⅡ联合消化，结合Percoll密度梯度离心可获取数量多、活性高的小鼠原代TEC。

ObjectiveTo compare the effects of several methods to isolate mouse primary thymus epithelial cells （TECs）, so as to screen out a simple, efficient, and practical method. MethodsMouse thymus tissues were digested using the following enzymes： collagenase Ⅰ （ColIE, 1 mg/ml）, dispase Ⅱ （DisⅡ, 3 mg/ml）, ColIE＋low-concentration DisⅡ （ColIE/DisⅡ-L, 0.75 mg/ml）, Col IE＋medium concentration DisⅡ （ColIE/DisⅡ-M, 1.5 mg/ml）, Col IE＋high-concentration DisⅡ （Col IE/DisⅡ-H, 3 mg/ml）, with or without Percoll density gradient centrifugation. Then, the number of cells were counted using a blood cell counter plate, and the cell viability was detected by trypan blue staining. The proportions of thymus stromal cells （TSCs） and TECs were detected by flow cytometry. ResultsCompared with enzyme digestion with Col IE or DisⅡ alone, a combination of Col IE and different concentrations of DisⅡ could rapidly digest thymus tissue （P〈0.01）, resulting in an increased number of thymus total cells （P〈0.05 or P〈0.01）, especially in Group Col IE/DisⅡ-M. Compared with enzyme digestion with Col IE or DisⅡ alone, the combination of Col IE and different concentrations of DisⅡ could present an increased proportion of TSC and TEC in Groups Col IE/DisⅡ-M and Col IE/DisⅡ-H （P〈0.01）. Furthermore, the proportions of TSC and TEC were improved after Percoll density gradient centrifugation （P〈0.05）. In addition, all the methods of isolation mentioned above could result in higher cell viability. ConclusionsBased on the combined use of Col IE and DisⅡ followed by Percoll density gradient centrifugation, mouse primary TECs can be obtained with higher cell viability.