阳离子聚合物介导的体外培养兔角膜内皮细胞基因转移

目的观察非病毒载体阳离子聚合物(SofastTM)介导的编码增强型绿色荧光蛋白(EGFP)的pEGFP-N1和pIRE-EGFP两种质粒对体外培养的兔角膜内皮细胞(RCEC)的转染效率和转染前后细胞Na+-K+-ATPase活性变化,探索最优的体外RCEC非病毒载体基因转移条件。方法消化法原代培养RCEC,SofastTM与pEGFP-N1和pIRE-EGFP两种质粒按不同比例混合,介导此两种质粒转染RCEC,分别比较各质粒不同时间的转染效率,并检测转染与未转染细胞Na+-K+-ATPase的活性以确定基因转移对RCEC活性的影响。结果SofastTM与质粒(pEGFP-N1和pIRE-EGFP)按不同比例转染RCEC后,24-48 h均有EGFP的表达。SofastTM∶pEGFP-N1=3.2∶1组在转染后48 h时转染效率最高(P<0.05),为3.6%。SofastTM∶pIRE-EGFP=3.2∶1组在转染后24 h时转染效率最高(P<0.05),为3.5%。转染与未转染组细胞数和细胞Na+-K+-ATPase活性均无明显差异。结论SofastTM可有效介导以pEG-FP-N1和pIRE-EGFP为载体的外源基因向体外培养的RCEC转移,且基因转移后RCEC活性不受影响。

Transferring gene to rabbit corneal endothelial cells in vitro by cationic polymer

Objective To observe the transfection efficiency of transferring plasmids pEGFP-N1 and pIRE-EGFP encoding enhanced green fluorescent protein(EGFP) to rabbit corneal endothelial cells(RCECs) by cationic polymer and the change of cell activity by detecting the activity of Na+-K+-ATPase.Methods RCEC was cultured by digestion and identification by neurone specific enolase(NSE) stain.Sofast TM mediated plasmid pEGFP-N1 and pIRE-EGFP to transfect RCECs and we compared the transfection efficiency at different time and in different groups by calculating the number of fluorescent cells.We observed the activity change of transfection and non-transfection RCEC by detecting the activity of Na+-K+-ATPase.Results After pEGFP-N1 and pIRE-EGFP transfected RCEC with Sofast TM by different ratio,RCEC expressed EGFP in 24-48 h.After transfection 48 h the group of SofastTM∶pEGFP-N1=3.2∶1 had the max transfection efficiency 3.6% and the max transfection efficiency of group SofastTM∶pIRE-EGFP =3.2∶1 was 3.5%,at 24 h after transfection.There were no obvious differences between the transfection and non-transfection groups of cell number and activity of Na+-K+-ATPase.Conclusion Sofast TM can mediate exogenous gene trasfering to RCEC effectively.pEGFP-N1 and pIRE-EGFP are the suitable non-virus vectors for gene transferring to RCECs.