Bone Marrow Mesenchymal Stem Cells Transfected With HHCN4 Genes By Lentivirus Can Be Modified To Be Biological Pacemaker In Vitro

Background:In the past 70 years, artificial ectrioc ardiac pacemaker has rescued many patients who sufferred with the heart block disease(sick sinus syndrome,atrial ventricular block) and intractable heart failure,ventricular fibrillation.However,gradually it snagged some shortage,such as limited batery life(8-10 years), the need for permanent catheter implantation into the heart, non-physiological excited procedure,lack of response to autonomic neurohumors and complications(infection,bleeding,myocardial perforation,loose electrode placement),and so on.Ideal pacemaker should be the"biological pacemaker",considenring phusiologic function of heart and adaptability of human body.Biological Pacemakers include gene therapy and cell therapy.Gene therapy has 3 kinds of strategies: (1) overexpression ofβ2-adrenergic receptors. (2) Inhibition of Kir2 gene family encoding inward rectifier current Ik1. (3) overexpression ofα(HCN2) andβ(MiRP1) subunit genes of inward depolarizing curent.There search in gene therapy have not get breakthrough.And the target genes of gene therapy are not pivotal moduIation gene of spontaneous depolarization of sinoatrial cells.Lots of studys indicated that,Funny Current (If), encoded by the hyperpolarization- activated cyclic-nucleotide-modulated (HCN1～4) channel gene family,plays a significant role in the process of spontaneous diastolic depolarization of sinoatrial cells. Especially,expression of HCN4 in sinoatrial node(SAN) is highest.Therefore, HCN4 is a optimal bioIogical pacemaker target gene because it is essential for modulation If and maintenance of electri-physiologic function of spontaneous cell in SAN.The bone mesenchymal stem cells not only have pluripotent differentiation potential,but aslo show high transfection and high stability under inducement of foreign gene.Many scholars used those characteres of MSCs as a platform for delievery of genes.The strategy take profits of cell engieneer and gene therapy.Obvious If-like current occur in mesenchymal stem cells(MSCs) transfected with mHCN2,and the vebtricular spontaneous rhythms got evident improvement after implantation of this MSCs in vivo.the physiological function of subtype HCN2 is to stabilize membrane potential in diastolic phase under quiescent condition,but fails to response to exercise and adrenergic nerve.While,the HCN4 not only play an important role in normal pacemaker potential and basal heart rate,but also participates in regulation of heart rate by adrenergic nerve.Objective:We adopted the hHCN4 as the target gene of biological pacemaker,utilized the platform of MSCs and obtained the MSCs that had stably high expression of hHCN4 through lentivirus vector transfection.Then we detected the expression of channel protein, and determine the kinetics characters of If channal in this MSCs transfected.The aim of this study was to establish a research base of the neotype and effective cell pacemaker therapy.Methods :1. Isolation and purification of swine mesenchymal stem cells were established by the density gradient centrifugation and the adherent method.Living cell morphology was observed by the invert microscope dynamically.2. hHCN4 lentivirus vector transfects MSCs: The hHCN4 lentivirus vector (Lentiv-EGFP- hHCN4) and the control vector(Lentiv-EGFP) respectively transfected the second filial generation MSCs.After transfected, the green fluorescent protein(GFP) was observed by fluorescence microscope and the expression of hHCN4 channel protein was detected by immunocytochemical stain.3. The expression of hHCN4 channel protein was detected by Western blot.4. Whole-cell patch clamp detected ionic currents kinetics of transfected hHCN4 and got density-voltage curves.Results:1. Under the invert microscope,most MSCs in primary culture were fibroblast-like spindle cells ,cloning growth.2. The hHCN4 lentivirus vector (Lentiv-EGFP- hHCN4) and the control vector (Lentiv-EGFP) respectively transfected MSCs successfully.and the cells stably express the gene of hHCN4.3. The expression and distribution of the Green fluorescent protein(GFP) were without the difference in the MSCs of two groups,observed by fluorescence microscope.Both vectors got 90% successful ratio of transfection on MSCs.4. After transfected, the HCN4 antibody stain was positive in group HCN4 cells, while weakly positive in group GFP and the control, in immunocytochemical stain.5. After transfected, the MSCs in group HCN4 were highly expressed hHCN4 channel protein (a strap about 170kDa), while weakly in group GFP and the control, in the Western blot.6. Whole-cell patch clamp recorded ionic currents of transfected hHCN4 and got density-voltage curves.The threshold for activation of IhHCN4 was -70±5mV. IHCN4 is blocked by 4mmol/L CsCl.The group GFP and the control has not IHCN4.Conclusion:1. The method of density gradient centrifugation Isolated and purificated swine mesenchymal stem cells.2. hHCN4 as a target gene of biological pacemaker,successfuly transfected MSCs by retrovirus vector, expressing hHCN4 channel protein.3. hHCN4 channel in transfected cell has the same kinetic characters as physiologic If current.