关键词: 牙龈, 上皮细胞, 原代细胞培养

Abstract:

Objective：To establish a stable primary culture method of human gingival epithelial cells, with a higher successful rate and shorter culture time. Methods: Nine patients who received “crown-lengthening surgery” with relatively healthy periodontal conditions were selected (n=9). Gingival samples were collected from the 9 donors during gingivectomy. Gingival epithelial cells were isolated and cultured by both an advanced enzyme digestion method and a tissue explant method. In the advanced enzyme digestion culture process, 2.5 g/L DispaseⅡwas used to separate the epithelial tissue part from the connective tissue part, which lasted for one night. Then the epithelial tissues were digested by 0.025% trypsin without EDTA for 10 minutes, and centrifuged by keeping the digested epithelial tissues that remained. This advanced method not only decreased the concentration and digesting time of the two above-mentioned enzymes, but also simplified the centrifugel process. The tissue explant method was not changed too much compared with the original method. Growing processes of the primary cells cultured by the two methods were observed and recorded respectively, and indirect immunocytochemical staining was used to identify the type of cultured cells. At the same time, successful rates and cell culture time were also compared between the two methods. Results: Human gingival epithelial cells with typical morphology could be cultured within a shorter period by the advanced enzyme digestion method with a successful rate of 88.9%, and proliferated rapidly as sheets. After 10－14 d cells could be passaged, gradually turned to be like fibroblasts when passaged to the third generation, and eventually went to apoptosis. The primary culture time was longer by using the tissue explant method, and approximately after 17－22 d cells could be passaged, although the successful rate was the same as the enzyme digestion method. Cytokeratin staining was both positive by indirect immunocytochemical staining of cells. Conclusion: Primary human gingival epithelial cells cultured by the advanced enzyme digestion method could grow faster and be passaged to the second generation successfully, which could supply a stable origin for cellular experiments.

Key words: Gingiva, Epithelial cells, Primary cell culture