目的构建Wnt-1重组腺病毒,并观察Wnt-1对人表皮干细胞分化相关蛋白表达的影响。方法连接穿梭质粒与骨架质粒,构建重组腺病毒;扩增后感染人表皮干细胞,检测其3种角蛋白和基质金属蛋白酶(MMP)-2的表达。结果重组质粒酶切后可得到30kb和4.5kb两条特异性条带;人表皮干细胞感染重组腺病毒后,聚合酶链反应(PCR)与绿色荧光蛋白均指示Wnt-1基因的表达,且角蛋白10表达变为阳性;角蛋白18表达增强;角蛋白19表达减弱;细胞培养基中MMP-2浓度由(-16.25±2.40)μg/L升高至(714.88±30.45)μg/L(t=58.45,P〈0.01)。结论Wnt-1具有促使人表皮干细胞向腺样上皮细胞分化的趋势。
Objective To construct recombinant adenovirus vector carrying Wnt-1 and observe its influence on differentiation of epidermal stem cells. Methods pAdTrack-CMV-Wnt-1 and pAdEasy-1 were linked to construct recombinant adenovirus vector. After the epidermal stem cells were infected with recombinant adenovirus,3 kinds of keratin and MMP-2 were detected. Results Recombinant adenovirus vector could be incised to 30 kb and 4.5 kb specificity straps. After epidermal stem cells were transfected with Wnt-1 gene, keratin10 expression became positive, keratin 18 expression was enhanced and keratin 19 expression reduced;Concentrations of MMP-2 were stepped up from ( -16.25 ± 2.40 ) μg/L to (714.88 ± 30.45) μg/L (t = 58.45, P 〈 0.01 ). Conclusion Wnt-1 could induce differentiation of epidermal stem cells into glandular epithelium-like cells.