Study On The Impact Of Lentivirus That Co-transfected By Human HCN4and Tbx3Gene On The Biological Activity

Objective：The limitation of electronic pacemaker encourages researchers to find a newway to cure bradycardia, one of methods is to rebuild a new cell of sinoatrial nodecell function. Now the main research is using HCN4to transfect stem cells, whichcan differentiate to the myocardial cells. As less of sinus mode maker gene, there isstill a gap between them. The gene of Tbx3exists in the sinoatrial node precursorcell during the later period of mature embryo, which retains the pacing function ofthe sinoatrial node precursor cell, and inhibits the sinoatrial node precursor cell to bedifferentiated. At the same time, TBx3is closely related to the occurrence of tumor,the overexpression of TBx3has been proved in breast cancer, carcinoma of bladderand hepatoma. The study aimed to construct the lentivirus particles thatsimultaneously bound with human HCN4gene, Tbx3gene and enhanced greenfluorescence protein (eGFP) gene. Based on this, the impact of HCN4and Tbx3onthe biological activity of mice embryonic stem cell was investigated, laying afoundation for the further study on how to rebuild sinoatrial node cell functionthrough the co-expression of HCN4and Tbx3.Methods：1. Packing the lentiviral plasmid and calculating the titerBy using the liposome approach, four types of targeting-gene-bound subjectplasmids (hHCN4-hTbx3-eGFP, hHCN4-eGFP, hTbx3-eGFP and eGFP) and a helperplasmid were packaged respectively. The293FT cells were co-transfected with thesefour types of plasmids respectively, which generate four types of resultant lentiviralplasmid. Finally, the number of fluorescence clone and the virus titer of each type ofco-transfected cells were then calculated.2. Sifting and transfecting of stem cellThe transduction of mice ES cells by the four types of resultant lentiviralplasmid were performed respectively, observing the effectiveness transfection underthe fluorescence microscope48hours later. Passage amplification the successfully transfected cells and pick cloning purification them. Through RT-PCR, theexpressions of the targeting genes were further evaluated.3. The evaluation of biologic activity of ES cellThe morphologies and the degrees of fluorescence expression of the four typesof resultant transgenic mice ES cells were observed. Then to determine the OD ofeach ES cells of MTT and depict its growth curve, to analyse the rate of cell apoptosisby the flow cytometer.Results:1. The construction of hHCN4-hTbx3lentiviralAfter the co-transfection of293FT cells with four types of plasmids, thefluorescence microscopic observation showed that all four types of lentivirus particles(hHCN4-hTbx3-eGFP, hHCN4-eGFP, hTbx3-eGFP and eGFP) were successfullyconstructed, The results of virus titer were4.7×10~7TU/ml,5.2×10~7TU/ml,4.5×10~7TU/ml and6.7×10~7TU/ml respectively.2. Detecting the expression if the related gene of Transgenic ES cellsAfter being transfected by four types of lentivirus, the fluorescence expressionsof ES cells upon picking and purification of the clones were normal. The results ofRT-PCR showed that the expressions of the targeting genes were moderate.3. The change of the apoptosis and morphology of ES cellsThe fluorescence expressions of ES cells upon picking and purification of theclones were normal. Among them, the cells in both hHCN4-eGFP and eGFP grouphad better cellular morphology and a relatively higher fluorescence rate. Floatingdead cells can be saw in the culture media supernatant by the flow cytometer andMTT, the rates of cell apoptosis in groups (hTbx3-eGFP, hHCN4-hTbx3) increasedrespectively, and the rates of growth in these groups were significantly slower.Conclusion:1. The lentiviral vectors that simultaneously bound with HCN4-Tbx3weresuccessfully constructed.2. The HCN4and Tbx3were successfully overexpressed in stem cells3. Tbx3appeared not to promote the proliferation of ES cell.