猪肺脏气道上皮干细胞的特异抗体筛选与抗原表达研究  

Selection of Specific Antibody and Expression of Stem Cell Markers in Porcine Airway Epithelial Cells

作　　者：杨佳丽[1] 石娟[1] 马凌洁 包少文[1] 郝秀静[1] 曾瑾[1] 李勇[1] 刘晓明[1] 

机构地区：[1]西部生物资源保护与利用教育部重点实验室,宁夏大学生命科学学院,宁夏银川750021

出　　处：《动物医学进展》2013年第9期47-52,共6页Progress In Veterinary Medicine

基　　金：国家自然科学基金项目(31172278,31260615)

摘　　要：猪肺脏气道上皮主要由基底细胞,克拉拉细胞,纤毛细胞,杯状细胞等上皮细胞类型组成,筛选猪气道上皮干细胞的相关特异性抗体,观察其特异抗原的表达,将有助于对猪气道上皮干细胞的分离、鉴定和生物学特性的研究。试验中,制备远端气道上皮细胞的冰冻切片与细胞爬片,利用免疫荧光染色法,分析不同物种来源的抗体对猪气道上皮细胞的免疫染色反应;同时利用Oct3/4、Sox2、CD133和SSEA-1等10种上皮干细胞相关抗体分析猪气道上皮干细胞表面抗原表达情况。通过试验,筛选出在猪气道上皮干细胞相关抗体,并发现气道组织冰冻切片未检测到这10种抗体相关的阳性细胞,而在细胞爬片中观察到CD49f和CD117阳性细胞数量较多。免疫荧光共染发现这些CD49f和CD117阳性细胞同时也表达基底细胞表面标记Keratin 14。这可能是正常情况下猪气道上皮干细胞数量极为稀少,表面抗原表达量低而不易检测到,在肺脏气道上皮受到外环境刺激时(如消化),上皮干细胞开始增殖以完成损伤修复作用发生数量上的增殖。综上所述,利用Keratin 14和CD49f或CD117(c-kit)双标染色法可以鉴定猪肺脏气道上皮干细胞的候选亚群来用于其生物学特性与功能的研究。The majority of cell types composed in the distal airway of porcine are basal cells, ciliated cells, goblet cells and Clara cells. The expressions of stem cell specific antigens and their specific antibodies were evaluated by an immunofluorescent staining in the present study. The distal airway epithelial cells were e- valuated for the expression of markers of epithelial cell types and stem cells by immunofluorescent staining on frozen sections or cytoslides using distinct specific antibodies. Immunofluorescent analysis showed no detectable expression of specific antigens for airway epithelial stem cells on the airway frozen sections when the antibodies against 10 potential stem cell markers （such as Oct3/4, Sox2 and SSEA-1） were utilized. However, the CD49f and CD117 were detected to co-express with Keratin 14 in a subset of epithelial cells on cytoslides. Only infrequent stem cells reside in the epithelium for detection at a stead state of lung. Up- on the injury （such as dissociation of epithelial cells）, the stem cells is able to undergo proliferation for in- jure repair, which in turn to elevate the expression of specific antigens of stem cells. Thus, it is a practica- ble approach using a combination of Keratin 14 and CD49f or CDl17 for stem cell sorting as double stem cells, by which the resulting subpopulation（s） of potential stem cells could be used for studies of cell biolo- gy and functions.

关 键 词：猪 肺脏气道上皮细胞 干细胞 表面标记抗原 免疫荧光染色 

分 类 号：Q26[生物学—细胞生物学]