HCN4转染大鼠骨髓间充质干细胞重建起搏离子通道I_f

Reconstruction of I_f channel in HCN4 transfected mesenchymal stem cells in rats

目的研究慢病毒载体介导超极化激活的环核苷酸门控通道基因4(HCN4)转染大鼠骨髓间充质干细胞(rMSCs)重建起搏离子通道的可行性。方法全骨髓法分离大鼠股骨及胫骨的MSCs,流式细胞术鉴定rMSCs;应用四质粒表达系统构建包含目的基因HCN4的慢病毒载体lentiV-HCN4-GFP,对照组慢病毒载体为仅含有报告基因GFP的慢病毒载体lentiV-GFP;慢病毒载体转染rMSCs;RT-PCR及荧光显微镜检测HCN4在rMSCs中的mRNA及蛋白表达;电压钳检测阳性转染细胞的起搏电流的特点。结果 HCN4基因转染MSCs的阳性率约60%;实验组RT-PCR产物出现480 bp的目的条带;阳性转染细胞中可记录到明显的时间和电压依赖性的超极化激活内向电流(If),激活的阈电压为-80 mV,该电流对低浓度CS+敏感;对照组未检测到相应的mRNA表达和超极化激活的电流。结论慢病毒载体能够介导HCN4基因高效转染MSCs,表达为起搏离子流通道。

Objective To reconstruct the If channel by transfection of hHCN4 gene in rat mesenchymal stem cells（rMSCs）.Methods Bone marrow was collected from SD rats by flushing femars and tibias with DMEM.The third passage cells were used for experiments.The identification of MSCs was carried out by flow cytometry when immune reaction with CD45 and CD90 antibodies.The lentivirus vector Lenti-GFP-HCN4 was constructed by plasmid pRsv-REV,pMDlg-pRRE,pMD2G and pCDH1-GFP-HCN4 in 293 cells.After transduced MSCs into lentivirus vectors,RT-PCR and immunofluorescence were used to detect the expression of mRNA and protein of hHCN4 channel.Finally recorded the If in the positively transfected cells with whole-cell voltage clamp.Results The transfection efficiency for MSCs was about 60%.There was hHCN4 mRNA in MSCs transfected with HCN4.The recorded hyperpolarization-activated inward current from positive MSCs was time-and voltage-dependent.The activated threshold voltage was about-80 mV.The current was sensitive to CsCl.The control cells didn＇t show the current at the same clamp voltage.Conclusion The functional If channels can be reconstructed in rMSCs overexpression with hHCN4.