Expression And Signifigance Of Bone Marrow Mesenchymal Stem Cells Transfected With Different Proportions Of HHCN1and HHCN4by Lentivirus

Background:Sick sinus syndrome is a common clinical disease,but the mechanism is yet to bedefined. In the present medical condition, we mainly use the permanent electronic pacemaker. Butwith the advantages come with the disadvantages, such as limited battery life, the need for permanentimplanation of electrodes into the heart and lack of response to nerve-humoral factors regulation.Lately, considering the physical function of heart and adaptability of human body, we bring upthe concept of the biological pacemaker. The related current, If,encoded by thehyperpolarization-activated cyclic-nucleotide gated, plays an important role in theprocess of spontaneous diastolic depolarization of sinoatrial cells. Therefore, we putforward bone marrow mesenchymal stem cells(MSCs) transfected with HCN as cellplatform to construct a “biological pacemaker” which provides experimental basis for thealternative to electronic pacemaker for clinical treatment in the future.Objective:We adopt the lentiv-GFP, carrying green fluorescent, as a vector to study thecharacteristics of the pacemaker current in the MSCs co-transfected with differentproportions of hHCN1and hHCN4. Meanwhile, we do some chemical tests on the cellstransfected positive aimed at the establishment of cell–genetically modified organismpacing therapy experimental basis in vitro.Methods：1. Extraction, culture and purification of SD rat MSCs.2. Lentiv-hHCN1and Lentiv-hHCN4transfect and co-transfect the MSCs in differentproportions.Lentiv-hHCN1and Lentiv-hHCN4in different proportions transfect the3rdgenerationof MCS, at the same time, we set hHCN1and hHCN4individually transfecting MSCs and lentiv without HCN gene as a control group.3.Detection of Lentiv-GFP-hHCN1and Lentiv-GFP-hHCN4transfection.Observe the expression of the green immumofluorescene with a fluorescenemicroscopy.Real-time quantitative polymerase chain reaction(RT-PCR) detection of the hHCN1and hHCN4expression.The expression of hHCN1and hHCN4protein detected by Western blot.4.We use whole cell patch clamp technique to record the pacemaker current in the cellstransfected with hHCN1and hHCN4.Results:1.Most primary cultured MSCs were fibroblast-like spindle cells in dispersedarrangement under ordinary invert microscope.2.The hHCN1/hHCN4transfect and co-transfect the MSCs successfully and each groupexpresses green fluorescent protein under a fluorescence microscope with a transfectionefficiency of about50-70%.3.RT-PCR confirmed the existence of mRNA in the positive transfected MSCs. We cansee bands around99kDa and150kDa in cells transfected with hHCN1and hHCN4inWestern blot.4. The whole cell patch clamp technique was used to record positive transfected MSCscurrent, and we got the voltage-dependence of hyperpolarization-activated inwardcurrent which activated at the voltage-75mV. In contrast, there was no pacemakercurrent in GFP group and the control group of MSCs. The current density in groupsco-transfected with hHCN1and hHCN4were larger than that of transfected with hHCN1and hHCN4individually.Conclusion:1.rat bone marrow mesenchymal stem cells can be successfully extracted and purifiedwith bone marrow adherent cell separation method.2.hHCN1and hHCN4by lentiviral vectors successfully transfected MSCs which expressed the corresponding channel protein and current which laid the foundation forgene therapy for arrhythmia.3.Positive transfected MSCs were able to express the characteristics of HCN channelsand generate the If which builded the foundation for constructing the biologicalpacemaker in vitro.