CAJ | 학술논문

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CC120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CC120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7.08×105转导单位(TU)/ml、1.88×105TU/ml和2.08×105TU/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.
목적 채용중조만병독CCL20기인특이성shRNA재체감염인영생화각질형성세포계(HaCaT),사선출은정간우CCL20기인표체적세포극륭병검측기간우효과.방법 용CaCl2법장이구건호적3충pHSER-CCL20-shRNA-GFP재체(pHCG-1화pHCG-2위CC120기인특이성,pHCG-3위CCL20기인착배)전염293FT포장출만병독과립,류식세포술측정기병독적도.용G418압력사선만병독감염적HaCaT,형광정량RT-PCR화ELISA법분별검측CC120기인mRNA화단백적표체수평,이판단기특이성간우효과.결과 삼충만병독재체소포장출적만병독적도분별위7.08×105전도단위(TU)/ml、1.88×105TU/ml화2.08×105TU/ml;감염후적HaCaT경과G418사선5～8주,획득4주CCL20기인특이성적세포극륭(HaCaT-1、HaCaT-2、HaCaT-3화HaCaT-4)급2주CCL20기인착배대조적세포극륭(HaCaT-5화HaCaT-6).경형광정량PCR화ELISA법검측현시,4주CCL20기인특이성세포극륭균구유은정간우효과,불부능현저지하조CCL20기인적mRNA표체(기억제솔분별위81.0%、89.0%、81.3%、77.2%),이차명현지감소CCL20단백분비(기억제솔분별위70.0%、86.1%、88.1%、90.7%).결론 채용중조만병독CCL20기인특이성shRNA재체감염HaCaT가이사선출구유장기은정표체RNA간우효응적CCL20기인고저형인영생화각질형성세포극륭,장가능위저면역배척반응이기인조직공정피부적구건제공충자세포.
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 supematants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively. Results The titers of three lentivimses were 7.08 ×105 transduced units(TU)/ml, 1.88×105 TU/ml and 2. 08 ×105 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 hath 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 keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shBNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.