【摘要】 目的 探讨E-cadherin对人脂肪干细胞(hADSCs)成表皮分化的影响。方法 利用胶原酶消化法从成人脂肪组织中分离培养hADSCs。携带增强型绿色荧光蛋白(EGFP)基因的慢病毒载体(Lv-EGFP)介导E-cadherin基因以10、25、50和100的感染复数(MOI)作用72 h转染hADSCs,荧光显微镜下观察荧光强度并统计转染效率。Lv-EGFP-E-cadherin过表达组慢病毒和Lv-EGFP对照组慢病毒分别转染hADSCs,未转染的hADSCs为空白组；4天后RT-PCR检测E-cadheirn mRNA的表达。将各组hADSCs经成表皮诱导14天后，RT-PCR检测表皮细胞标志物CK18、ZO-1 mRNA的表达情况。结果 3代以后hADSCs呈均一长梭形，增殖活跃。MOI=10、25、50、100时，EGFP阳性率分别为51.9%±2.68%、95.1%±3.82%、95.6%±2.56%、96.9%±2.25%,选择MOI=25为最佳转染滴度进行后续实验。与对照组及空白组相比，转染Lv-EGFP-E-cadherin过表达慢病毒可显著增强hADSCs中E-cadherin mRNA的表达(P<0.05),E-cadherin过表达后可显著增高细胞中CK18、ZO-1 mRNA的表达(P<0.05),促进成表皮分化。结论 E-cadherin过表达可在一定程度上促进hADSCs向表皮细胞诱导分化。更多还原

【Abstract】 Objective To explore the effects of E-cadherin over-expression on the epidermal differentiation of human adipose-derived stem cells(hADSCs). Methods hADSCs were isolated from adult liposuction tissue by collagenase digestion. Then, lentivirus vectors carrying green fluorescence protein EGFP(Lv-EGFP) mediated E-cadherin gene to be transfectd into hADSCs at different values of MOI(10, 25, 50 and 100, respectively) for 72 hours. The fluorescence intensity and the rate of transfection were observed by fluorescence microscopy. The lentiviruses in the Lv-EGFP-E-cadherin over-expression or Lv-EGFP control groups were transfected into hADSCs, while untransfected hADSCs were set as the blank group. After four days, the expression of E-cadherin mRNA was determined by RT-PCR. Then, hADSCs in each group were induced to differentiate towards epidermal cells for 14 days. Then, the expression of epidermal cell markers CK18 and ZO-1 were measured by RT-PCR. Results The hADSCs grew actively, with a long spindle shape. The positive rate of EGFP was 51.9%±2.68%, 95.1%±3.82%, 95.6%±2.56%, 96.9%±2.25% when MOI was 10, 25, 50, and 100, respectively. The optimal value of MOI was set at 25 for the following tests. Compared with the control and blank groups, the levels of E-cadherin mRNA in hADSCs was remarkably enhanced by lentiviruses in the Lv-EGFP-E-cadherin over-expression group(P<0.05). Moreover, E-cadherin overexpression could significantly increase the amounts of CK18 and ZO-1 mRNA to stimulate epidermal differentiation(P<0.05). Conclusion The E-cadherin over-expression can promote the epidermal differentiation of hADSCs towards epidermal cells.更多还原