摘要
目的:建立高效、稳定的人扁桃体隐窝上皮细胞原代培养方法。方法:收集80例3~5岁儿童手术切除的扁桃体组织,分别采用组织块贴壁法和Ⅱ型中性蛋白酶联合0.25%胰蛋白酶-EDTA消化法提取扁桃体隐窝上皮细胞并比较两种方法的提取效果;应用无血清角化细胞培养基进行细胞纯化及原代培养;用相差显微镜观察细胞形态和生长特点、免疫细胞化学染色及免疫荧光技术鉴定细胞来源。结果:Ⅱ型中性蛋白酶联合消化法在分离扁桃体隐窝上皮细胞成功率、细胞密度以及细胞融合时间方面均高于组织块贴壁法(P<0.05),细胞接种后第2天开始贴壁,外观呈现多边形,岛状生长,培养12 d左右,细胞连成单层,呈现铺路石样生长。免疫细胞化学染色显示广谱角蛋白表达阳性,波形蛋白阴性;免疫荧光鉴定角蛋白8/18染色阳性。结论:应用Ⅱ型中性蛋白酶联合0.25%胰蛋白酶-EDTA消化法以及无血清角化细胞培养基可建立高效稳定的人扁桃体隐窝上皮细胞原代分离方法和体外培养体系。
Objective：To establish stable and efficient method for primary culture of human palatine tonsil crypt epithelial cells.Methods：Eighty palatine tonsil tissues were derived from children aged from 3 to 5 undergoing surgical removal of tonsil. Tissue piece method and combined type Ⅱ dispase and 0.25% trypsin-EDTA digestion method were used to separate primary palatine tonsil crypt epithelial cells,and the effectiveness of two isolation methods were compared. Serum-free Keratinocyte medium were applied to purification and primary cultivation. Growth feature and morphology of primary cells were observed under an inverted microscope.Immunofluorescence and immunocytochemistry technique were used to identify the specification of primary cell. Results：The success rate,density and primary cell fusion time of the dispase digestion group were all higher than those of the tissue piece group（P〈0.05）.The primary tonsil crypt epithelial cell began adherent growth after 2 days isolation from the tonsil tissues. The shape of cell was polygon under microscope,and cells connected like islands. Primary cells formed confluent monolayers after 12 days＇ cultivation,it seemed like paving stone. Pancytokeratin positive staining was determined by immunocytochemistry. The specific cytokeratin 8/18 staining was positive after immunofluorescence identification. Conclusion：It is an efficient and repeatable method for primary isolation and cultivation of human palatine tonsil crypt epithelial cells by using combined type Ⅱ dispase and 0.25% trypsin-EDTA digestion method with serum-free keratinocyte medium.
作者
邢娇
黄群
李琦
金玉
Xing Jiao1, HuangQun2, Qun2, Li Qi2 ,Jin Yu1(1Department of Gastroenterology, 2Department of Otolaryngology , the Affiliated Children＇ s Hospital of NMU , Nanjing 210008, Chin)
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2018年第3期294-298,342,共6页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(81672020)
