The Experimental Study Of Tbx18 Transfection Inducing Mice Cardiac Stem Cells Differentiate Into Pacemaker-like Cells

Objective:Through increasing the expression of Tbx18 in mouse cardiac stem cells by adenovirus mediation,to investigate whether that can induce mouse CSCs differentiate into pacemaker-like cells in vitro.Methods:1.Mouse CSCs culture and separation:Take SPF grade Kunming mice heart tissue to obtain primary mouse CSCs through cardiac tissue adherent culture,until the CSCs were grown to approximately 80%confluence,differential digestion was performed to separate and collect CSCs,then identified by immunofluorescence of C-kit⁺.2.Experimental groups:the primary CSCs were divided into three groups:（1）blank control group:only primary CSCs;（2）negative control group:CSCs infected with adenovirus without target gene;（3）Tbx18 experimental group:CSCs infected with adenovirus carrying Tbx18 gene.3.Construction of Tbx18 over-expression vector:Tbx18 plasmid DNA as the template for PCR amplification,recombinant plasmid vector underwent gene sequencing,and verification by gene sequence comparison with the target gene sequence.4.Packaging of Tbx18 over-expression adenovirus and titer measurement:Using the constructed Tbx18 over-expression vector to transfect HEK293T cells for packaging the result of packaging was tested by Western-blot detection,then collected and amplified virus for virus titer measurement.5.The verification of the feasibility of Tbx18 infecting CSCs.The growth and morphological changes of CSCs were record after infection with Tbx18 adenovirus,and the expression of Tbx18 in mouse CSCs was detected by Western-blot.6.Verification of up-regulating Tbx18 induced CSCs to differentiate into pacemaker-like cells:CSCs infected with Tbx18 over-expression adenovirus were cultured for 6 days,qPCR and Western blot were used to detect the expression of HCN4 in mouse CSCs in mRNA level and protein level.Results:1.Primary mouse CSCs were obtained through cardiac tissue adherent culture method:cardiac tissue began to adhere to the wall from 4th hour after dish planking.The tissue adhered firmly to the wall after 1～2 days and the fibroblasts grew around the tissue.In from the 5th to the 7th day,small,round,bright cardiac stem cells began to grew around cardiac tissue,they grew in upper layer of the fibroblasts,and the nucleoli couldn’t be observed under high magnification;in the13th to the15th day while the cells grew to about 80%confluence,which were separated and collected for C-kit⁺immunofluorescence identification.Five views were randomly selected in fluorescence microscope,GFP positive cells percentage of the total number under vision was accounted about 75%,so the C-kit⁺CSCs was about 75%of the total cells.2.Construction of Tbx18 over-expression vector was successfully completed,and gene sequencing results proved the gene sequence was completely accordance with the expected sequence;Green fluorescent expression was observed in the second day after HEK293T cells were infected by Tbx18 over-expression vector,the fluorescence intensity reached highest level in 10th～12th day.The virus liquid was collected,and the value of virus titer was 1.0×10¹⁰IFU/ml.Western-Blot testing showed Tbx18 protein highly expressed in the HEK293T cells.3.In the process of CSCs culture after being infected by Tbx18recombinant adenovirus,most of them underwent non-typical morph-ology changes,showing spindle or irregular shape,and there were no significant difference between Tbx18 experimental group and blank control group or negative control group.Green fluorescence expression was visible on 24th hour after CSCs were transfected by Tbx18 recombined adenovirus,the fluorescence intensity reached highest level on 72th hour.MOI value was 800.Western blot detected 69KD size Tbx18 protein expression in Tbx18 experimental group,but there was no Tbx18 protein expression in both blank control group and negative control group.4.On the 6th day after Tbx18 infection,there was no HCN4expression in mRNA level and protein level in the blank control group,negative control group and Tbx18 experimental group tested by qPCR and Western Blot.Conclusions:1.Obtaining of mouse CSCs through tissue adherent culture method was stable and reliable.C-kit⁺CSCs was about 75%of the total number of cells.2.Construction of Tbx18 recombined adenovirus vector was successfully completed.3.Tbx18 recombined adenovirus was transfected to CSCs successfully,MOI value was 800.4.Up-regulation of Tbx18 expression in CSCs could not induce CSCs differentiate into pacemaker-like cells.