免疫荧光双标法在小鼠表皮干细胞分子标记筛选中的应用  

Screening of molecular markers for epidermal stem cells in mice by the double immunofluorescence labeling technique

作　　者：陈萌[1] 任宁[2] 刘文君[1] 秦晓敏[1] 李劲松[1] 

机构地区：[1]山东大学齐鲁医院病理科,济南250012 [2]济南市第一人民医院病理科,济南250011

出　　处：《山东大学学报（医学版）》2011年第4期65-69,共5页Journal of Shandong University:Health Sciences

摘　　要：目的用标记滞留细胞(LRCs)及免疫荧光双标记法进行小鼠表皮干细胞可靠分子标记的筛选。方法选择出生5 d的昆明小乳鼠进行腹腔注射5-溴,2-脱氧尿嘧啶(5-bromodeoxyuridine,BrdU),隔日注射,连续7 d。于注射后第90天进行取材,在冰冻切片上分别进行BrdU与整合素β1、P63和CD34的双重免疫荧光标记,观察这些标记物与BrdU的符合度。结果 90 d后小鼠皮肤上皮细胞中仍有少数阳性细胞,主要存在于毛囊的隆突部位,另有少数阳性细胞散落在基底层。免疫荧光双标的结果示,Brdu与整合素β1双标阳性的细胞占所有整合素β1阳性细胞的81%,占所有Brdu阳性细胞的94%;Brdu与P63双标阳性的细胞占所有P63阳性细胞的65%,占所有Brdu阳性细胞的86%;Brdu与CD34双标阳性的细胞占所有CD34阳性细胞的30%,占所有Brdu阳性细胞的62%。结论整合素β1与BrdU的符合率最高,P63其次。β1和P63是这些分子标记物中筛选干细胞最好的指标。Objective To screen reliable molecular markers for epidermal stem cells（ESCs） in mice by using the double immunofluorescence labeling technique and label-retaining cells（LRCs）.Methods The epidermis was labeled for 7 days with 5-bromodeoxyuridine（BrdU） in postnatal day 5（P5） mice,followed by a trace period of up to 12 weeks.The tissue of the mice was frozen and sliced in a frozen microtome after 90 days.Slices were stained with polyclonal antibodies of anti-β1 integrin,P63 and CD34 and monoclonal antibodies of anti-Brdu,and then observed under a fluoroscope.Results After 90 days,few LRCs were found in a region of the outer root sheath adjacent to the insertion of the arrector pili muscle,known as the bulge region.The percentage of double immunofluorescence cytochemically stained β1 integrin-and BrdU-positive cells was 81% in all β1-positive cells,and 94% in all BdrU-positive cells.The percentage of P63-and BrdU-positive cells was 65% in all P63-positive cells,and 86% in all BdrU-positive cells.The percentage of CD34-and BrdU-positive cells was 30% in all CD34-positive cells,and 62% in all BdrU-positive cells.Conclusion Among these molecular markers,β1 integrin,with highest accordance with BrdU,is the best molecular marker for ESCs,and P63 is the next best.

关 键 词：标记滞留细胞 表皮干细胞分子标记 免疫荧光双标 

分 类 号：R322.99[医药卫生—人体解剖和组织胚胎学]