不同时段iv胶原黏附表皮干细胞的生物学研究-第三军医大学学报.docVIP

表皮干细胞差速分选的研究方法 崔洪勋，伍津津，唐书谦，杨桂红，李元朝（第三军医大学大坪医院野战外科研究所皮肤科，重庆 400032） 摘 要：目的 观察不同时间内表皮干细胞对IV型胶原的粘附特性，为表皮干细胞的分选提供实验依据。方法 分别取贴壁后10、20、30、60 min后的人角质形成细胞，用细胞计数器进行细胞计数，计算其直径。并用β1 整合素、K19 对贴壁的细胞进行鉴定。流式细胞仪测β1整合素的阳性表达量，相差显微镜进行细胞形态学观察。结果 随着时间的延长，表皮干细胞贴壁数逐渐增多，20 min内粘附细胞数与10 min内比较差异有统计学意义(P0.05)，而与30 min、60 min内比较无统计学意义。贴壁细胞小而圆，反光能力强，整合素β1间接免疫荧光免疫组化显示贴壁细胞均为阳性，K19免疫组化阳性率为31.03%，流式细胞仪测整合素β1阳性率表达量100%。结论 20 min内IV胶原能快速粘附绝大部分表皮干细胞，细胞活力不受影响，可以用于表皮干细胞的分选。 关键词：表皮干细胞；IV型胶原；细胞分选 中图法分类号： 文献标识码：A Research approach of sorting epidermal stem cells within different time by adhering to type IV collagen CUI hong-xun，WU Jin-jin, TANG shu-qian, YANG Gui-hong, LI Yuan-chao (Department of Dermatology ,Daping Hospital ,Research Institute of Field Surgery, Third Military Medical University of Chinese PLA , Chongqing 400042, China ) Abstract: Objective To observe the adhere characteristic of epidermal stem cells onto IV collagen at different time , in order to provide a laboratory evidence to sort the epidermal stem cell .Methods keratinocytes were isolated from adult human foreskins circumcision and inoculated on the six-well dish coated IV collagen ,counted the cells and measured the size of diameter with a CASY at 10 min 、20min、 30min、 60min ,and the expression of integrin-β1 with FCM, identified with integrin-β1 and CK19 by immunofluorescence and immunocytochemical. Morphological observe by contrast phase microscope. Results morphologically: epidermal stem cell are smaller and rounder, reflect strongly, adhering populations at 20minis higher than 10 min (P0.05), having statistically significant, expression of integrinβ1 by FCM is 100%, Conclusion: Most of epidermal stem cells can be sorted by adhering to IV collagen at 20min, however highly pure epidermal stem cells need further investigation. Key words: epidermal stem cells IV collagen cell sorting 表皮干细胞对基底膜的粘附性是维持干细胞特性的基本条件。目前,体外分离、纯化表皮干细胞也是利用干细胞对细胞外基质的粘附性进行的。Kim等把分离出的角质形成细胞按照粘附到IV型胶原的能力分为快速粘附细胞、慢速粘附细胞和非粘