不同时段IV胶原黏附表皮干细胞的生物学研究.docVIP

基于对IV型胶原在不同时间内粘附特性的分选表皮干细胞方法的研究 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 Sorting epidermal stem cells on basis of adhering to type IV collagen within different times CUI hong-xun, WU Jin-jin, TANG shu-qian, YANG Gui-hong, LI Yuan-chao (Department of Dermatology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China ) Abstract: Objective To observe the adhesive property of epidermal stem cells onto IV collagen at different time points, in order to sort the epidermal stem cells. Methods Keratinocytes were isolated from adult human foreskins circumcision and inoculated on the 6-well plates precoated with IV collagen. The cell number was counted and the diameters were measured with a CASY at 10, 20, 30 and 60 min after implanting. The expressions of integrin β1 and CK19 on the adhered cells were examined with immunofluorescence and immunocytochemical methods to identify epidermal stem cells. The expression of integrin-β1 was observed with flow cytometry. Morphological observation was carried out with the aid of contrast phase microscope. Results With the elapse of time, more and more cells adhered, and the number of those adhering within 20 min was significantly larger than those within 10 min (P0.05). Morphologically the epidermal stem cells were brighter, smaller and rounder after the adhesion. All adhered cells were positive to integrin-β1 and had a positive rate of 31.03% to CK19. FCM indicated that the cells were 100% positive to integrin β1, Conclusion: Most of e