ABSTRACT
The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied.The cultured MSCs were transfected with pIRES2-EGFP plasmid carrying enhanced green fluorescent protein (EGFP) gene and mHCN2 gene.The identification using restriction enzyme and sequencing indicated that the mHCN2 gene was inserted to the pIRES2-EGFP.Green fluorescence could be seen in MSCs after transfection for 24-48 h.The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot,and the quantity of mHCN2 mRNA and protein expression in transfected MSCs was 5.31 times and 7.55 times higher than that of the non-transfected MSCs respectively (P＜0.05,P＜0.05).IHCN2 was recorded by whole-cell patch clamp method.The effect of Cs+,a specific blocker of pacemaker current,was measured after perfusion by patch clamp.The results of inward current indicated that there was no inward current recording in non-transfected MSCs and a large voltage-dependent inward and Cs+-sensitive current activated on hyperpolarizations presented in the transfected MSCs.IHCN2 was fully activated around -140 mV with an activation threshold of-60 mV.The midpoint (V50) was -95.1±0.9 mV (n=9).The study demonstrates that mHCN2 mRNA and protein can be expressed and the currents of HCN2 channels can be detected in genetically modified MSCs.The gene-modified MSCs present a novel method for pacemaker genes into the heart or other electrical syncytia.