人永生化角朊细胞表达干细胞表型的流式细胞术分析

Stem Cell Phenotypic Analysis of Human Immortal Keratinocyte Line HaCaT by Flow Cytometry

观察人永生化角朊细胞系HaCaT细胞表达的几种干细胞表型,为以该细胞系构建人组织工程皮肤和进行基因操作提供相应的实验依据。方法:采用无血清培养基对HaCaT细胞进行培养,获取处于对数生长期的细胞进行直接荧光双标法、直接荧光单标法和间接荧光单标法以分别CD49fCD71、角蛋白K19、CD29和CD133,依托流式细胞仪检测上述抗原在HaCaT细胞的表达情况。结果:特异性较高的角朊干细胞表型CD49f+D71-和K19+在HacaT细胞的百分率分别为16．6±2．8%和14．94±1．23%。CD29+HaCaT细胞为49．55±6．68%,可能包括了角朊干细胞和短暂扩增细胞。分别反映细胞黏附特性和增殖能力的CD49f+HaCaT细胞和CD71+HaCaT细胞各占98．1±0．8%和82．1±3．9%。HaCaT细胞仅表达3．43±0．77%的干细胞表型CD133。结论:人永生化角朊细胞系HaCaT细胞的表型特征表明它具有较高的角朊干细胞和短暂扩增细胞比例以及很强的黏附基底膜特性和增殖能力,提示它可以作为构建人组织工程皮肤种子细胞和基因修饰角朊干细胞的替代物。

Objective： To observe several stem cell phenotypes of human immortal skin keratinocyte line （HaCaT） and provide experimental evidence for applying these cells to engineered human skin and gene modification. Methods ：The HaCaT cells were cultured in defmed keratinocyte-serum free medium （DK-SFM） and harvested in log phase growth period. The relevant stem cell like phenotypes were labeled by immunofluorescence, ie. direct dual labeling for GD49f and GD71, direct singular labeling for CD29, indirect labeling for GD133 and K19. The percentages of these phenotypes were analyzed by flow cytometry. Results： Either CD49f^＋CD71^- or K19^＋ phenotype, more specific marker than others for keratinocyte stem cells, had similar percentage in HaCaT cells, ie. 16.6＋2.80/0 and 14.94±1.230/0, respectively. For the marker of keratinocyte stem cell and transient ampliflcating cell, the percentage of CD29＋ HaCaT cells was 49.55±6.680/0. 98.1±0.80/0of CD49f^＋HaCaT cells and 82.1±3.90/0of CD71^＋HaCaT cells reflected the ability of adhesion and proliferation, respectively . The GD133 had only 3.43±0.770/0 in HaCaT cells . Conclusion：The phenotypic characteristics of human immortal skin keratinocytes （HaCaT） indicate that they have higher percentages of keratinocyte stem cell and much stronger capacities of adhesion and proliferation than normal human keratinocytes, which suggests that HaCaT cells would be used as a substitute of keratinocyte stem cells for fabricafin, g engineered skin and manipulating epidermis relevant genes.