Rats Mesenchymal Stem Cells Modified By MHCN2 Gene To Create Biologic Pacemakers

Background Abnormalities of impulse generation and propagation induce cardiacarrhythmias. Although electronic pacemakers are currently the mainstay of therapy for heartblock and other electrophysiological abnormalities, they are not optimal. Among theirshortcomings are limited battery life, the need for permantent catherter implantation intothe heart, and lack of response to autonomic neurohumors. For these reasons, several genetherapy approaches have been explored as potential alternatives.Hyperpolarization-activated cyclic nucleotide-gated channel (HCN) played animportant role in the automatic depolarization of the diastolic sinus node. HCN gene familyhave 4 members, HCN1-HCN4. HCN2 is an optimal biological pacemaker target genebecause it is essential for modulation funny current and maintenance of electric-physiologicfunction of pacemaker cells in sinus node. Self-proliferation and multipotency, two majoradvantages of stem cells, makes its research and application one of the hot spots in themedical and biological research activities at home and abroad. Where, embryonic stem cellsof the capacity to differentiate into all the tissues in the organism have drawn the mostattention. In spit of the tremendous potential in the medical applications, ethic controversiesarose due to the constraints its material sources. In recent years, studies found that morethan the capacities to differentiate into tissue-specific cells, adult stem cells can also have multi-lineage, multi-layer differentiation of embryos. In addition, they can differentiate intoother cells or tissues, creating broader applications of stem cells. Adult stem cells also canget easy access to material to avoid tissue typing and immunity rejection, easy for importand expression of gene. We selected rats mesenchymal stem cells, one kind of the adultstem cells, as the vector of genetic transmission, and tried to use transgenic technology toestablish the biological pacemaker, in attempt to replace electronic pacemakers in theapplication of clinical treatment.Objective This experiment was executed using selected the rats mesenchymal stemcells modified by gene HCN2 as the target. The pacing current expressed by us constructthe plasmid pIRES2-EGFP-mHCN2 carrying a gene HCN2 and markers, and transferredthe plasmid to MSCs by liposome. Gene HCN2 was detected by the expression of nucleicacid, protein, and the current level, providing the evidences on the feasibility of testing forbiological pacemaker and mesenchymal stem cells as gene transfer vectors.Methods MSCs were obtained by density gradient centrifugation method andadherence separation then identified by flow cytometer. The plasmid pGH-mHCN2 andplasmid pIRES2-EGFP were digested by EcoRâ… and BamHâ… . The objective fragmentswere reclaimed and linked by T4 DNA Ligase. The recombinant plasmid was transformedto the competent cells and chose the masculine colony on the next day. Restriction enzymeand sequencing method were used to proof that mHCN2 was insert to pIRES2-EGFP. Theobjective gene was transfected with Lipofectamine 2000 into MSCs and the transfectingresults were observed by fluorescence microscope. The expression of mHCN2 mRNA andprotein in the transfected cells were identified by RT-PCR and Western blot. I_HCN2 wasrecorded by whole-cell patch clamp. The effect of Cs⁺ which is the specificity blocker ofpacemaker current on I_HCN2 was detected.Results MSCs were proved correctly by flow cytometer and the purity was 95%above. The identification using restriction enzyme and sequencing indicated that themHCN2 was inserted to the pIRES2-EGFP. The green fluorescence can be seen intransfected MSCs after 24 to 48 hours under fluorescence microscope. The mHCN2 mRNAin transfected MSCs is 5.31 times of the MSCs by RT-PCR (P＜0.05). The mHCN2 protein in transfected MSCs is 7.55 times of the MSCs by Western blot (P＜0.05). Non-transfectedMSCs demonstrated no significant voltage-dependent currents, mHCN2-transfected MSCsexpressed a large voltage-dependent inward current activating on hyperpolarizations. I_HCN2was fully activated around -140 mV with an activation threshold of -60 mV. The midpoint(V₅₀) was (-95.1±0.9) mV. Cs⁺ (4 mmol/L) obviously blocked the current.Conclusion We success construct the plasmid pIRES2-EGFP-mHCN2 and make itexpress in MSCs by Lipofectamine 2000.We demonstrate that mHCN2 gene can express inmRNA and protein levels in MSCs. mHCN2-transfected MSCs expressed the currents ofphysiological pacemaker current character. Its may substitute the sino-atrial nodepacemaker cells and play important effects in depolarization.