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目的探讨4种不同方法体外分离、培养小鼠阴道粘膜干细胞(VMSCs)所得结果的差异,确定最适合体外培养VMSCs的方法。方法选择组织块培养法、胰酶消化培养法、胶原酶消化培养法、胰酶和胶原酶混合培养法对小鼠VMSCs进行原代培养,并通过HE染色、SP免疫组化法鉴定VMSCs,分析4种方法的优劣。结果 4种培养法中,胶原酶消化培养法细胞生长很差,无法得到VMSCs;组织块培养法培养时间较长,培养出的细胞包含成纤维细胞较多;单纯胰酶消化培养法,可获取较单一的VMSCs,但细胞活性相对较差;只有胰酶、胶原酶混合培养法,在适当的比例和浓度时,可发挥最佳作用,获取数量多、较纯的VMSCs。且经HE染色、免疫组化法鉴定结果均显示培养出的细胞即是VMSCs。结论 4种方法中有3种体外分离培养VMSCs的方法,均可得到VMSCs,其中以胰酶、胶原酶混合培养法效果最佳。
OBJECTIVE: To investigate the differences of the results of in vitro isolation and culture of mouse vaginal mucosal stem cells (VMSCs) by four different methods to determine the most suitable method for culturing VMSCs in vitro. Methods VMSCs were primarily cultured in tissue culture, trypsinization, collagenase digestion, trypsin and collagenase co-culture. VMSCs were identified by HE staining and SP immunohistochemistry. The merits of the four methods. Results In the four culture methods, collagenase digestion and culture showed poor growth of cells, and VMSCs could not be obtained. Tissue explant culture method took longer to culture and contained more fibroblasts. Pure trypsin digestion culture method could obtain Compared with single VMSCs, the cell activity was relatively poor. Only trypsin and collagenase mixed culture method could get the best effect and obtain more and more pure VMSCs at appropriate ratio and concentration. After HE staining, the results of immunohistochemistry showed that the cultured cells were VMSCs. Conclusion VMSCs can be obtained from VMSCs isolated and cultured in vitro using three of the four methods. Among them, trypsin and collagenase were the best.