摘要
目的探讨多效生长因子基因(pleiotrophin,Ptn)转染小鼠脂肪来源干细胞(adipose-derived stem cells,ADSCs)作为组织工程血管的重建种子细胞的可行性,并检测其基因表达,为缺血性病损治疗提供新手段。方法取3只清洁级近交系C57BL/6W雌性小鼠(体重15～20 g)腹股沟皮下脂肪,采用酶消化法体外分离、培养ADSCs,流式细胞仪行细胞表面标志物CD29、CD44鉴定。实验分为转染pIRES2-LEGFPN1组(含Ptn基因编码序列,A组)和转染pLEGFP-N1负对照组(含GFP基因编码序列,B组)。ADSCs转染后,A组以最佳筛选浓度200μg/mL的G418对转染后细胞进行筛选,流式细胞仪鉴定转染后细胞免疫表型。实时荧光定量PCR、Western blot检测A、B组细胞Ptn基因和PTN蛋白表达。结果实验成功分离、培养小鼠ADSCs;细胞表面标志物CD29、CD44阳性率分别达99.5%、95.8%,双阳性率达93.6%。A组转染Ptn后细胞表面标志物CD29、CD44阳性率分别达99.1%、95.6%,双阳性率达93.4%。实时荧光定量PCR、Western blot检测示,A组Ptn基因和PTN蛋白相对表达水平均显著高于B组(P<0.05)。结论 Ptn基因能转染小鼠ADSCs并高表达PTN蛋白,为组织工程血管构建种子细胞选择提供了新思路。
Objective To study the transfection and expression of pleiotrophin（Ptn） gene in mice adipose-derived stem cells（ADSCs） so as to provide a new approach for the treatment of ischemic injury. Methods ADSCs from clean inbred C57BL/6W mice（weighing,15-20 g） were isolated and cultured in vitro.The cell surface markers（CD29 and CD44） of ADSCs were identified by flow cytometry.The ADSCs were transfected with plasmid pIRES2-LEGFPN1（containing Ptn gene coding sequence） as experimental group（group A） and with plasmid pLEGFP-N1（containing GFP gene coding sequence） as control group（group B）.After ADSCs were transfected by different plasmids respectively,the cells containing Ptn gene were selected by G418（the best selected concentration was 200 μg/mL）,and the immunophenotype of the cells was identified by flow cytometry after transfection.Meanwhile,real-time fluorescence quantitative PCR and Western blot were used to analyse the expression levels of Ptn mRNA and PTN protein in selected cells. Results The mice ADSCs were isolated and cultured successfully in vitro.The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.5% and 95.8%,respectively;the double positive rate of CD44 and CD29 was 93.6%.The positive rates of the cell surface markers CD29 and CD44 of ADSCs were 99.1% and 95.6%,respectively after transfection of Ptn gene;the double positive rate of CD44 and CD29 was 93.4%.The expression levels of Ptn gene and PTN protein in group A were significantly higher than those in group B（P 0.05）. Conclusion The ADSCs can be stablely transfected by Ptn gene,the transfected ADSCs can express PTN protein highly,which is a new idea for tissue engineering of vascular reconstruction.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2013年第8期897-901,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
四川省科技厅科技支撑计划项目(4160220)~~
