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目的比较体外培养条件下人原代表皮干细胞与传代细胞的生物学差异。方法选择2009年5-12月在我科医学美容门诊行包皮环切术后的包皮标本共20例,从包皮标本获得人原代表皮干细胞,利用0.25%胰蛋白酶冷消化4 h获得传代细胞,利用CASY系统计数后计算细胞的黏附率,倒置相差显微镜下观察原代干细胞及传代细胞的形态学变化,观察干细胞表面标志物整合素β1、CK19,细胞分化的表面标志物CK10的表达,以及各代细胞基因组的表达差异。结果原代干细胞的黏附率为(55.44±2.42)%,随着传代次数的增加,2代、4代及6代细胞的黏附率分别为(44.30±2.76)%、(30.95±2.24)%、(20.64±1.37)%,与原代细胞相比,差异有统计学意义(P<0.05)。细胞生长融合时间随着传代次数而增加。各代细胞均表达整合素β1;原代、2代及4代细胞表达CK19,但6代细胞不表达CK19;部分6代细胞表达CK10。原代及4代细胞基因组比较结果相近,但与6代细胞结果差异较大。结论与原代干细胞相比,体外培养的人表皮干细胞在4代以后生物学特性发生改变,体外实验宜选用4代以前的细胞。
Objective To compare the biological differences between human primary epidermal stem cells and passage cells cultured in vitro. Methods From May to December in 2009, 20 cases of foreskin circumcision were performed in our medical cosmetic clinic. Human primary epidermal stem cells were obtained from foreskin specimens, and passaged cells were obtained by cold digestion with 0.25% trypsin for 4 h. The cell adhesion rate was calculated after CASY system counting. Morphological changes of primary stem cells and passage cells were observed under inverted phase contrast microscope. The expression of integrin β1, CK19, the surface marker of cell differentiation CK10 and the expression of CK19 Differences in the expression of cell genomes. Results The adherence rate of primary stem cells was (55.44 ± 2.42)%. The attachment rates of the 2nd, 4th and 6th generation cells were (44.30 ± 2.76)% and (30.95 ± 2.24)%, respectively, (20.64 ± 1.37)%, respectively. Compared with the primary cells, the difference was statistically significant (P <0.05). Cell growth and fusion time increased with the number of passages. All the cells expressed integrin β1; primary, second and fourth generation cells expressed CK19, but the sixth generation did not express CK19; some of the sixth generation cells expressed CK10. The results of the primary and fourth generation cell genomes are similar, but the results of the sixth generation cells are quite different. Conclusion Compared with the primary stem cells, the human epidermal stem cells cultured in vitro changed their biological characteristics after the 4th generation. In vitro experiments, the cells prior to passage 4 should be selected.