整合素β1对人表皮干细胞增殖与分化的影响

Influence of integrin β1 on the proliferation and differentiation of human keratinocyte stem cells

目的构建针对整合素（intergrin）β1特异性的siRNA表达载体，并转染人表皮干细胞（KSC），探讨整合素β1对KSC增殖与分化的影响。方法针对目的基因intergfinβ1，选择RNA干扰作用靶点特异性的寡核苷酸序列，构建到siRNA表达质粒pSilencer3．1／H1中，并转染人KSC，转染分组为转染siIntegrinβ1组、非转染组、转染空载体组、转染siNegative组。通过蛋白质印迹法观察intergrinβ1及外皮蛋白的表达变化；应用流式细胞仪观察细胞周期变化；挑选干细胞克隆传代培养14d后用1％罗丹蓝染色计算克隆形成率。结果重组载体siIntegrinβ1转染人KSC能够抑制intergrinβ1的蛋白表达，抑制效率达70％；KSC中外皮蛋白表达较对照组增加50％；G0／G1期细胞较对照组明显减少（P〈0．01）、G2期及S期细胞较对照组增多（P〈0．05）；干细胞克隆形成率较对照组显著降低（P〈0．01）。结论重组载体转染人KSC能下调intergfinβ1的表达，下调intergrinβ1的表达可能是表皮干细胞走向分化的重要调控因素之一。

Objective To construct small interfering RNA （siRNA） expression vectors specific to integrin β1 and study the role of integrin β1 in regulating human keratinocyte stem cell （KSC） proliferation and differentiation in vitro. Methods DNA oligonucleotides targeting integrin β1 were synthesized and inserted into pSinencer3.1/H1 plasmids. Then the siIntegrinβ1 was transfected into the KSCs. The KSCs were divided into control （non transfection）, TI （with transfection of vacant vector）, T2 （with transfection of siIntegrinβ1 ）, T3 （wlth transfection of siNegative） groups. The expression of integrin β1 and involucrin protein was detected by Western blotting. Cell cycle was analyzed by flow cytometry. The colony forming efficiency （CFE） of KSCs was determined by 1% rhodanile blue staining. Results Transfection of recombinant siIntegrinβ1 vector into KSC could inhibit the expression of integrinβ1 protein,with inhibition rate being of 70%. The protein expression of involucrin after transfection was increased by 50%, but the number of cells in G0/G1 stage was decreased （P 〈 0.01 ）, while that in G2 and S stage increased （ P 〈 0.05 ） as compared with that in controls. The CFE of KSCs after transfection was obviously lower than that in controls （ P 〈 0.01 ）. Conclusion Protein expression of integrin β1 in KSCs can be down-regulated by recombinant siIntegrinβ1 vector transfection. Integrin 131 might be one of the important factors in regulating differentiation in human KSCs.