慢病毒介导E-cadherin过表达对人脂肪干细胞成表皮分化的影响

Effects of lentivirus - mediated E - cadherin over expression on the epidermal differentiation of human adipose- derived stem cells

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72h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hAD-SCs为空白组；4天后RT-PCR检测E-cadheimmRNA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-1mRNA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-cadherinmRNA的表达（P〈0．05），E-cadherin过表达后可显著增高细胞中CK18、ZO-1mRNA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。

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 colla- genase 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 fluo- rescence intensity and the rate of transfection were observed by fluorescence microscopy. The lentiviruses in the Lv - EG- FP- 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 expres- sion 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 fol- lowing 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 to- wards epidermal ceils.