The Study Of Transcription Factor TBX18 Promoting Rat Bone Mesenchymal Stem Cells To Biological Pacemaker Cells

Backgruond:A large number of diseases are induced by malfunction of the cardiac pacemaker and acknowledged as "sick sinus syndrome".The syndrome often require the implantation of electronic pacemakers.Nevertheless,this method results in many disadvantages,like battery failure,lead repositioning,infections and other complications.These handicap necessitate developing biological pacemaker.The formation of biological pacemaker can come from differentiation of pluripotent stem cells(PSCs)or by reprogramming.The SAN is the pacemaker of the heart.Transcription factor TBX 18-expressing mesenchymal progenitors in the flow tract region that differentiate into pacemaker myocardium to form the SAN.It is indicated that the head and tail represent separate regulatory domains expressing distinctive gene programs.BMSCs have various advantages compared with the rest of stem cells.It is all known that BMSCs have the superiority to differentiate into various types of cells in vitro.On account of these characteristics,MSCs are usually used as a carrier for transmitting biological pacemaker genes to lead the formation of functional and phenotypic pacemaker cells.Objective:This experiment is applied to construct the TBX18 adenovirus vector which infecting adult rat BMSCs to biological pacemaker cells.Methods:1.The isolation,purification and characterization of BMSCs Bone marrow were come form adult male Sprague-Dawley(SD)rats,BMSCs were isolated by the whole bone marrow adherent method,then the cells were analyzed by flow cytometry,to observe cell morphology by invert microscope.2.The construction of human TBX18 gene adenovirus vector Firstly,we should construct recombinant TBX 18 adenovirus vector and sequence.By using LipofiterTM approach,targeting gene plasmid pHBAd-MCMV-GFP-TBX18 and the helper plasmid pHBAd-MCMV-GFP were packaged separately,the HEK 293 cells were co-transfected with above two plasmids,and then amplified.The revised TCD50 method was used to detect the virus titer of each plasmid.3.Human TBX18 gene adenovirus vector transfects BMSCs The TBX18 adenovirus vector(pHBAd-MCMV-GFP-TBX18)and the control vector(pHBAd-MCMV-GFP)transfected the P3-P5 BMSCs respectively.After transfected,the green fluorescent protein(GFP)was observed by inverted fluorescence microscope.Quantitative Real-time PCR was used to detect the expression of hTBX18 mRNA,Western blotting was used to detect the expression of hTBX18 protein,to ensure whether the gene TBX18 is successfully transfected into BMSCs.4.The detection of biological pacemaker markers RT-qPCR and Western blott:ing was used to detect the expression of biological pacemaker markers like ?-actin,cTnI,HCN4 and CX43.Results:1.To identify the phenotypes of BMSCs,we used flow cytometry.The results showed that BMSCs expressed stromal cell markers CD29 and CD90(up to 97%-99%),while there was almost no expression of the hematopoietic marker CD45.Which suggested the cells used here were purified BMSCs.The whole bone marrow adherent method can isolate the higher purity of BMSCs,BMSCs within 3-5 passages were used in the following experiment.2.The fluorescence microscopic observation showed that the target gene was constructed successfully.The virus titer of Ad-TBX18 was 1×1010PFU/ml.3.hTBX18 adenovirus vector can be transferred into BMSCs successfully,the hTBX18 mRNA and protein expressions can be detected by Western blotting and RT-qPCR(P<0.05).4.The expression of a-actin,cTnI and HCN4 gene was significantly higher in BMSCs infected with TBX18 adenovirus compared with the GFP groups and blank groups(P<0.05).While the expression of CX43 was significantly lower than the GFP groups and blank groups(P<0.05).