人角朊干细胞体外筛选及鉴定的实验研究

目的探讨体外筛选及鉴定人角朊干细胞(KSC)的方法. 方法利用KSC对细胞外基质的快速黏附性,选择不同时间黏附的细胞分为5、20和60分钟3组,并以不进行筛选的细胞作对照组.体外培养扩增后,以KSC的相对特异性标识分子β1整合素、角蛋白19(Ck19)的单克隆抗体,免疫组织化学方法对各组细胞进行鉴定,利用图像分析系统测平均灰度值.并用流式细胞仪、透射电镜对各组细胞进行检测. 结果各组细胞在10～14天均有克隆形成,组间比较5分钟组细胞生长较慢,但形成克隆较大,细胞体积较小.免疫组织化学β1整合素、Ck19在各组都有表达,各组平均灰度值统计学分析显示,β1整合素在5分钟组的表达与另3组间有统计学意义(P<0.05),Ck19的表达在5分钟组与20分钟组间无差异(P>0.05),60分钟组与对照组间差异不显著,而前两组与后两组之间比较有统计学意义(P<0.05).流式细胞仪检测提示5分钟组细胞大部分处于G1期,与其它3组有区别.透射电镜显示5分钟组细胞体小,核浆比大,细胞器不成熟. 结论利用细胞外基质筛选的5分钟组细胞处于幼稚状态,并有强克隆形成能力,符合预期的KSC特点,可利用KSC对细胞外基质的快速黏附性对其纯化筛选,同时利用β1整合素、Ck19的单抗加以鉴定.

SELECTION AND IDENTIFICATION OF HUMAN KERATINOCYTE STEM CELLS IN VITRO

OBJECTIVE: To investigate the selection and identification of human keratinocyte stem cells(KSC) in vitro. METHODS: According to the characteristics of KSC which can adhere to extracellular matrix very fast, we selected 3 groups of different time(5 minutes, 20 minutes and 60 minutes) and unselected as control group. And the cells were identified by monoclone antibody of beta 1-integrin and cytokeratin 19 (Ck19), then the image analysis was done. Furthermore we analyzed the cultured cells with flow cytometer(FCM) and observed the ultrastructure of the cell by transmission electron microscope(TEM). RESULTS: The cell clones formed in all groups after 10 to 14 days, while the cells of 5 minute group grew more slowly than those of the other groups, however, the clones of this group were bigger. The expression of beta 1-integrin and Ck19 were found in all groups. The positive rate of beta 1-integrin was significant difference between 5 minute group and the other groups (P < 0.05). And the expression of Ck19 was no significant difference between 5 minute group and 20 minute group(P > 0.05), and between 60 minute group and control group. But significant difference was observed between the former and the later groups(P < 0.05). The result of FCM showed that most cells of the 5 minute group lied in G1 period of cell cycle, which was different from those of the other groups. At the same time, the cells of 5 minute group were smaller and contained fewer organelles than those of the other groups. CONCLUSION: The above results demonstrate that the cells of 5 minute group have a slow cell cycle, characteristics of immaturity, and behaving like clonogenic cells in vitro. The cells have the general anticipated properties for KSC. So the KSC can be selected by rapid attachment to extracellular matrix and identified by monoclone antibody of beta 1-integrin and Ck19.