CCL20基因敲低型组织工程皮肤种子细胞的克隆筛选及其干扰效果

Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissueengineered skin

目的采用重组慢病毒CCL20基因特异性shRNA载体感染人永生化角质形成细胞系（HaCaT），筛选出稳定干扰CCL20基因表达的细胞克隆并检测其干扰效果。方法用CaCl2法将已构建好的3种pHSER—CCL20-shRNA—GFP载体（pHCG-1和pHCG-2为CCL20基因特异性，pHCG-3为CCL20基因错配）转染293FT包装出慢病毒颗粒，流式细胞术测定其病毒滴度。用G418压力筛选慢病毒感染的HaCaT，荧光定量RT—PCR和ELISA法分别检测CCL20基因mRNA和蛋白的表达水平，以判断其特异性干扰效果。结果三种慢病毒载体所包装出的慢病毒滴度分别为7．08×10^5转导单位（TU）／ml、1．88×10^5TU／ml和2．08×10^5TU／ml；感染后的HaCaT经过G418筛选5～8周，获得4株CCL20基因特异性的细胞克隆（HaCaT-1、HaCaT-2、HaCaT-3和HaCaT-4）及2株CCL20基因错配对照的细胞克隆（HaCaT-5和HaCaT-6）。经荧光定量PCR和ELISA法检测显示，4株CCL20基因特异性细胞克隆均具有稳定干扰效果，不仅能显著地下调CCL20基因的mRNA表达（其抑制率分别为81．0％、89．0％、81．3％、77．2％），而且明显地减少CCL20蛋白分泌（其抑制率分别为70．0％、86．1％、88．1％、90．7％）。结论采用重组慢病毒CCL20基因特异性shRNA载体感染HaCaT可以筛选出具有长期稳定表达RNA干扰效应的CCL20基因敲低型人永生化角质形成细胞克隆，将可能为低免疫排斥反应异基因组织工程皮肤的构建提供种子细胞。

Objective To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line （HaCaT）. Methods The three pHSER-CCL20-shRNA-GFP vectors （pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control） have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lectiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively. Results The titers of three lentiviruses were 7.08 × 10^5 transduced units（TU）/ml, 1.88 ×10^5 TU/ml and 2. 08 × 10^5 TU/ml, respectively. Two HaCaT cell clones from each lectiviral vectors were obtained after G418 screening for 5-8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly. Conclusions The four human immortal keratinoeyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.