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目的观察不同时间内表皮干细胞对Ⅳ型胶原的黏附特性。方法分别取贴壁后10、20、30、60min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。流式细胞仪检测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,20min内黏附细胞数与10min内比较差异有统计学意义(P<0.05),而与30、60min内比较差异无统计学意义。贴壁细胞小而圆,反光能力强,整合素β1间接免疫荧光及免疫组化结果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阳性率表达量为100%。结论20min内Ⅳ胶原能快速黏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。
Objective To observe the adhesion of epidermal stem cells to type Ⅳ collagen in different time. Methods Human keratinocytes were harvested at 10, 20, 30 and 60 min post-transplanta- tion, respectively. The cell counts were counted with a cell counter and their diameters were calculated. And β1 integrin, K19 adherent cells were identified. The positive expression of β1 integrin was detected by flow cytometry, and the cell morphology was observed by phase contrast microscopy. Results With the extension of time, the adherent number of epidermal stem cells gradually increased. The number of adherent cells within 20 min was significantly different from that within 10 min (P <0.05), but no significant difference with 30 and 60 min. The adherent cells were small and round with strong reflex ability. The results of indirect immunofluorescence and immunohistochemistry of integrin β1 showed that the adherent cells were positive, the positive rate of K19 immunohistochemistry was 31.03%, the positive rate of integrin β1 by flow cytometry The expression level is 100%. Conclusion Ⅳ collagen can rapidly adhere to most epidermal stem cells in 20min, cell viability is not affected, and can be used for the sorting of epidermal stem cells.