关键词: 骨髓间充质干细胞, 慢病毒, HCN, If

Abstract: 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 with Lentivs 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 -80mV. 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.

Key words: mesenchymal stem cells, Lentivirus, hyperpolarization-activated cyclic nucleotide-gated channel, If