上皮细胞条件培养液对BMSCs分化的影响  

EFFECTS OF EPITHELIAL CELL CONDITIONED MEDIUM ON DIFFERENTIATION OF BMSCs

作　　者：杨朋[1] 魏人前[1] 谭波[1] 李秀群[1] 王佳[1] 周昆鹏[1] 左潇[1] 李顺[1] 解慧琪[1] 

机构地区：[1]四川大学华西医院生物治疗国家重点实验室干细胞与组织工程研究室,成都610041

出　　处：《中国修复重建外科杂志》2009年第5期612-616,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：教育部新世纪优秀人才支持计划(NCET-07-575);国家自然科学基金资助项目(30670734);国家高技术研究发展计划(863)资助项目~~

摘　　要：目的探讨上皮细胞条件培养液(epithelial cell conditioned medium,ECCM)体外诱导犬BMSCs向上皮细胞分化的可行性。方法成年健康雄性比格犬1只,体重10kg。于犬髂后上棘采用骨髓穿刺法取骨髓5mL,分离培养BMSCs。取犬口腔黏膜下组织,剪成约4mm×4mm大小,制备ECCM。取第2代BMSCs以2×104个/孔细胞密度接种,实验组于ECCM中培养,对照组于低糖DMEM完全培养基中培养。观察两组细胞形态特征,MTT法绘制生长曲线,诱导培养21d免疫组织化学染色鉴定上皮细胞特异性标志物——细胞角蛋白19(cytokeratin19,CK-19)和细胞角蛋白AE1/AE3,透射电镜观察细胞超微结构。结果倒置相差显微镜下见两组细胞均呈长梭形,均匀分布,折光性较强,增殖迅速。两组细胞生长曲线均呈"S"形,实验组生长曲线较对照组右移,提示ECCM对细胞增殖有抑制作用。实验组CK-19和细胞角蛋白AE1/AE3免疫组织化学染色均呈阳性反应,对照组呈阴性。透射电镜下实验组细胞胞浆内可见紧密连接。结论ECCM体外有诱导同种BMSCs向上皮细胞分化的作用。Objective To investigate the feasibility of inducing canine BMSCs to di erentiate into epithelial cells in vitro with epithelial cell conditioned medium（ECCM）.Methods Five mL BMSCs were obtained from iliac spine of a healthy adult male canine with weighing 10 kg, and then isolated and cultured.The oral mucosa was harvested and cut into 4 mm × 4 mm after the submucosa tissue was eliminated;ECCM was prepared.BMSCs of the 2nd passage were cultured and divided into two groups, cultured in ECCM as experimental group and in L-DMEM as control group.The cell morphological characteristics were observed and the cell growth curves of two groups were drawn by the continual cell counting.The cells were identi ed by immunohistochemical staining through detecting cytokeratin 19（CK-19） and anti-cytokeratin AE1/AE3 on the 21st day of induction.The ultra-structure characteristics were observed under transmission electron microscope.Results The cells of two groups showed long-fusiform in shape and distributed uniformly under inverted phase contrast microscope.The cell growth curves of two groups presented S type.The cell growth curve of the experimental group was right shifted, showing cell proliferation inhibition in ECCM.The result of immunohistochemical staining for CK-19 and anti-cytokeratin AE1/AE3 was positive in the experimental group, con rming the epithelial phenotype of the cells;while the result was negative in the control group.The cells were characterized by tight junction under transmission electron microscope.Conclusion The canine ECCM can induce allogenic BMSCs to di erentiate into epithelial cells in vitro.

关 键 词：组织工程 BMSCS 上皮细胞 诱导分化 犬 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]