Wnt-1基因对人表皮干细胞的分化与增殖的影响研究

The effect of Wnt-1 gene on human epidermal stem cell differentiation and proliferation

目的探讨Wnt-1基因对人表皮干细胞分化与增殖的影响。方法将Wnt-1基因用重组腺病毒基因导入技术导入人表皮干细胞内,在证实Wnt-1基因在人表皮干细胞中导入成功后,检测人表皮干细胞的增殖能力、标记性分子、基质金属蛋白MMP-2、MMP-7浓度及细胞内Ca2+浓度等指标的变化。结果分离获取的人表皮干细胞在接种后第3天进入快速生长期,第7天时到达平台期;Wnt-1基因导入人表皮干细胞后,可观察到绿色荧光蛋白的表达,RT-PCR检测到了Wnt-1mRNA的存在,台盼兰excell系统检测发现人表皮干细胞直径无明显变化,而细胞增殖则明显加强;培养基中的MMP-2的浓度由感染前(-21.33±3.34)增加到感染后的(812.33±41.26)、MMP-7的浓度均值由感染前(17.33±2.31)增加到感染后的(1126.44±13.56),差异均有统计学意义(P<0.01);人表皮干细胞内Ca2+浓度明显升高,作为上皮终末分化标志的K10表达变为阳性;在汗腺分泌部表达的K18表达增强;作为干细胞和腺上皮细胞共同标记的K19的阳性明显减弱。结论导入Wnt-1基因后,人表皮干细胞增殖能力明显增强,细胞直径则没有明显变化;人表皮干细胞培养基中NPVIP-2和MMP-7的浓度均显著增高;Wnt-1的导入能够诱使人表皮干细胞内Ca2+浓度升高;Wnt-1基因具有诱使人表皮干细胞向腺样上皮细胞分化的倾向。

Objective To discuss the effect of Wnt-i gene on human epidermal stem cell differentiation and proliferation. Methods with recombinant gene technology, the Wnt-1 gene was recombinated into human epidermal stem cells. After confirming the recombination of Wnt-1 gene, the Wnt-1 protein expression in human epidermal stem cells before and after human epidermal stern cell proliferation, labeled molecules, matrix metalloproteinases MMP-2, MMP-7 concentration and intracellular Ca2＋ concentration were observed. Results After 3 days inoculation, separated human epidermal stem cells obtained a fast growing, and reached a plateau at day 7. Human epidermal stem cells combined with Wnt-1 gene were observed in green fluorescent protein expression, Wnt-1 mRNA existence was also detected by RT-PCR. These double standards testified the presence and expression of Wnt-1 gene in human epidermal stem cells. Trypan blue excell testing system found no significant changes in diameter of human epidermal stem cells, but cell proliferation was significantly enhanced. MMP-2, MMP-7 concentrations were significantly increased in the Medium, indicating that human epidermal stem cells obtained the molecular basis of degrading the extracellular matrix; Ca2 ＋ concentration significantly increased in human epidermal stem cells, and had the tendency of getting away from the ＂niche＂ of the stem cell; K10, as a sign of epithelial terminal differentiation, showed positive expression. K18, which expresses in the sweat gland, was found high expression. On the other hand, the expression of K19, which was co-labeled in stem cells and epithelial cells, significantly reduced. Conclusion Wnt-1 gene has the tendency of inducing human epidermal stem cells to differentiate into adenoid epithelial cells.