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 trans...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.展开更多
文摘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.
