改良人牙龈上皮细胞原代培养方法

目的：建立稳定的人牙龈上皮细胞的原代培养方法，为牙周组织的细胞学研究提供可靠的细胞来源，对原有的原代培养方法进行了改良，期望达到培养成功率高、原代培养时间短且操作简便的效果。方法： 选择行“冠延长术”的牙周相对健康者9人，取冠延长术切除的领圈牙龈组织进行牙龈上皮细胞的原代培养，培养方法包括改良酶消化法和组织块法。改良酶消化法使用2.5 g/L DispaseⅡ酶（Ⅱ型分离酶）浸泡组织块过夜，将上皮与结缔组织分离，再用0.025%（质量分数）不含EDTA（乙二胺四乙酸）的胰蛋白酶静置消化上皮碎片10 min，不弃去上皮碎片直接离心并重悬细胞，不仅降低了酶的消化浓度，减少了消化时间，还简化了重悬细胞的过程；组织块法相对以往方法并无改良。观察两种方法所培养原代细胞的生长过程，在培养成功后对细胞进行免疫细胞化学染色鉴定，并比较两种方法的成功率和培养时间。结果： 改良后的酶消化法能够比较快地培养出细胞特征明确的人牙龈上皮细胞，成功率达88.9%，且细胞成片状生长，10~14 d后可传代，传至3代后细胞形态逐渐不规则直至凋亡，而组织块法原代培养成功率虽然相同，但时间更长，17~22 d后才可传代。经免疫细胞化学染色，角蛋白染色阳性。结论： 改良酶消化法能够快速培养出原代人牙龈上皮细胞并传代，可作为细胞学研究的稳定细胞来源。

An modified culture method of primary human gingival epithelial cells

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 sam-ples were collected from the 9 donors during gingivectomy.Gingival epithelial cells were isolated and cul-tured 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 con-nective tissue part,which lasted for one night.Then the epithelial tissues were digested by 0.025% tryp-sin without EDTA for 10 minutes,and centrifuged by keeping the digested epithelial tissues that re-mained.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 morpholo-gy 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.Cy-tokeratin staining was both positive by indirect immunocytochemical staining of cells.Conclusion:Pri-mary 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 ex-periments.