人结合上皮细胞体外培养与冻存方法的改良  

The modification of culture and cryopreservation methods for human junctional epithelium cells

作　　者：李树波[1] 李德懿[1] 葛锡锐[2] 

机构地区：[1]上海交通大学医学院附属第九人民医院,上海市口腔医学重点实验室,上海市口腔医学研究所,上海200011 [2]中国科学院上海生命科学研究院细胞资源中心,上海200032

出　　处：《上海口腔医学》2007年第3期263-267,共5页Shanghai Journal of Stomatology

基　　金：国家自然科学基金(30672314);上海-联合利华研究与发展基金

摘　　要：目的:改良人结合上皮(junctional epithelium,JE)细胞的分离培养及冻存方法。方法:取正畸拔除的牙周组织健康的前磨牙,沿龈缘剪去冠方1mm左右的沟内上皮,用无菌11号刀片紧贴牙面刮下、剪碎JE组织,简化培养操作步骤。取第2代JE细胞,加入冻存液,按设定的降温程序(从0℃开始,以每分钟下降1.5℃速度下降至-18℃,保持5min;再以每分钟下降20℃的速度降至-80℃,然后投入-196℃液氮罐)冻存细胞,40℃水浴复苏,进一步研究JE细胞的形态、增殖、鉴定及复苏后存活率等生物学活性。结果:不用Dispase冷消化,采用0.1%胰蛋白酶-0.02%EDTA消化,同样可成功地培养人JE细胞;JE细胞复苏后的存活率为(93.87±3.11)%,生长状况良好,与第2代细胞相似;免疫细胞化学染色显示,培养的JE细胞角蛋白-19(cytokeratin-19,CK19)表达强阳性,而波形丝蛋白(vimentin)表达也为阳性。结论:改良的JE分离培养与冻存方法可行,JE表型在体内和体外并非完全一致,可能与细胞生长的底物有关。PURPOSE： To study the methods for isolating, culturing and cryopreserving junctional epithelium（JE） cells. METHODS： JE tissue samples were obtained from human periodontally healthy premolars which were extracted for orthodontic reasons. The sulcular epithelium was removed at a distance of 1 mm from the gingival margin. JE tissue was detached from the extracted teeth by using 11# sterile blade and minced into small fragments. The cultured JE cells were incubated with a freezing medium consisting of 90% calf serum and 10% DMSO. The freezing protocol was applied using a computer-controlled freezer to freeze the medium to -80℃ before cells were plunged into liquid nitrogen and stored. The above procedures were optimized to further study the morphology, proliferation, determination of JE cells and measure the viability after thawing. RESULTS： 0.1% trypsin containing 0.02% ethylenediamine tetraacetate （EDTA） digestion was used to isolate JE cells successfully without dispase. The viability of thawing JE cells was （93.87±3.11）%, and the morphology was similar to that of the second passage JE cells. CK19 and vimentin were both positive in JE cells immunocytochemically. CONCLUSION： The methods could be applied into practice. The phenotype of JE cells in vitro is not always consistent with that in vivo. It might be associated with the substrate which cells grow in contact with. Supported by National Natural Science Foundation of China （Grant No.30672314） and Research and Development Foundation of Shanghai Unilever.

关 键 词：结合上皮 细胞培养 细胞冻存 免疫细胞化学 

分 类 号：R780.2[医药卫生—口腔医学]