兔口腔黏膜细胞与灭活的3T3成纤维细胞的体外共培养  

Coculture of rat oral keratinocytes with inactive 3T3 fibroblasts

作　　者：李超[1] 徐月敏[1] 宋鲁杰[1] 崔磊[2] 尹烁[2] 

机构地区：[1]上海交通大学第六人民医院泌尿外科,上海200233 [2]上海交通大学上海组织工程研究与开发中心,上海200237

出　　处：《上海交通大学学报（医学版）》2007年第11期1297-1300,共4页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(30772159)~~

摘　　要：目的探讨口腔黏膜细胞的培养方法,为进一步以口腔黏膜细胞为种子细胞构建组织工程化尿道提供实验依据。方法体外分离口腔黏膜细胞,取部分细胞与经丝裂霉素灭活的3T3成纤维细胞进行共培养（共培养组）,定期观察细胞形态变化及生长增殖情况。以非共培养的口腔黏膜细胞作为对照（非共培养组）。同时,对体外培养获得的口腔黏膜细胞进行广谱角蛋白抗体（AE1/AE3）和K19免疫荧光染色鉴定;流式细胞仪检测第2代共培养组细胞的AE1/AE3阳性细胞百分比。结果共培养组口腔黏膜细胞,呈现典型的＂铺路石＂状,细胞形态均一,可传代至7-8代。非共培养组口腔黏膜细胞则形态多样,传至第2代时,即开始出现老化,细胞贴壁及增殖能力均明显减弱。免疫荧光染色显示,体外培养获得的口腔黏膜细胞AE1/AE3免疫荧光染色阳性,40%细胞K19染色阳性。流式细胞仪检测结果表明,第2代共培养组口腔黏膜细胞AE1/AE3阳性细胞百分比〉95%。结论口腔黏膜细胞在有灭活的3T3成纤维细胞存在时可成功培养和扩增,为进一步将其作为种子细胞构建组织工程化尿道奠定了良好的基础。Objective To explore the cultural method of oral keratinocytes and make preparations for further investigation in using oral keratinocytes as a new choice of seed cells for the reconstruction of tissue-engineered urethra. Methods Oral keratinocytes of rabbits were isolated in vitro and seeded onto a culture dish with a feeder layer of 3T3 mouse fibroblasts inhibited by mitomycin （i3T3） or a culture dish without i3T3 respectively. Cell morphology was observed and cell growth was detected at intervals. Meanwhile, oral keratinocytes obtained from in vitro culture were performed immunofluorescence staining with broad-spectrum keratin antibody （AE1/AE3） and keratin 19 antibody （ K19）. The percentage of positive cells of passage 2 reactive to AE1/AE3 was assessed by flow cytometry. Results Oral keratinocytes seeded onto a feeder layer of i3T3 exhibited finer morphous, better amplification capability, and could be passed for 7 or 8 generations. However, those cultured without i3T3 took on various morphous and could only be subcuhured 2 generations before ageing. It was indicated by immunofluorescence staining that oral keratinocytes obtained from in vitro culture were positive for AE1/AE3 staining and 40% were positive for K19 staining. The result of flow cytometry revealed that the amour of positive keratinocytes reactive to AE1/AE3 was more than 95% of total cellular score. Conclusion Oral keratinocytes of rabbits can be cocuhured with i3T3 in vitro and magnitude quantity can be attained, laying a favourable foundation for oral keratinocytes as a new choice of seed cells for urethral reconstruction with tissue engineering.

关 键 词：口腔黏膜细胞 3T3细胞 尿道 组织工程 中文摘要 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]