The Construction Of Thymosin β4Eukaryotic Expression Vector And Alpha Myosin Heavy Chain Promoter Driven Reporter Vector

Objectives: The aim of this master thesis:1) To construct thymosin β4eukaryoticexpression vector for gene therapy in myocardial infarction;2) To construct α-myosinheavy chain promoter driven reporter which is used as tracing marker to monitorcardiac differentiation of adult stem cells;3) To investigate the suitable dosage of thecardiac vessel specific peptide—MI1which is applied as cell face modificationtargeting molecule in bone marrow mesenchymal stem cells.Method:1) The pBlast49-, pcDNA3.0-Flag-and pIRES2-eGFP-vector were appliedto construct three kinds of thymosin β4eukarytic cell expression vectors respectivelyand they were transfected into293T,293F and HUVEC to observe the expression oftransduced thymosin β4gene via Real-time PCR, Western Blot and ELISA;2)Cardiac specific differential reporter vector was constructed via recombination ofα-myosin heavy chain promoter and eGFP as α-MHC-eGFP. The tracing effect forcardiac differentiation of constructed α-MHC-eGFP vector was investigated viatransducing into neonatal rat cardiomyocytes. In addition, the constructedα-MHC-eGFP vector was transduced into bone marrow mesenchymal stem cells first,and then induced cardiac differentiation to further tracing effect for cardiacdifferentiation which is confirmed as GFP and cardiac type III troponin I (TNNI3)immunofluorescence;3)The cardiac vessel specific peptide—MI1(50,250,500,1000,1500μg/mL) was applied to surface modification for bone marrow mesenchymal stem cells.The adhesion, proliferation and in vivo targeting of MI1modified bone marrow mesenchymal stem cells were investigated via cell culture andtail vein injection.Results:1) The DNA sequencing confirmed that the inter sequence in all threeconstructed expression vectors are matched to thymosin β4gene sequence totally.Real-time PCR analysis documented that the mRNA levels of thymosin β4in allconstructed thymosin β4expression vectors transduced cells were up-regulatedsignificantly. And the fluorescence intensity of all pIRES2-Tβ4-eGFP transfectedpositive cells was higher significantly than that of non-transduced control cells Inaddition, the Western Blot analysis for Flag tagged antibody (ligated downstream ofthymosin β4gene and co-expressed with thymosin β4gene) revealed that there was aFlag tagged positive band. It suggested that the transduced thymosin β4gene wasexpressed in mRNA and protein level;2) The green fluorescence positive cells werefound in constructed α-MHC-eGFP promoter vector transduced neonatal ratcardiomyocytes. In addition, some of GFP and cardiac type III troponin I positivecells were found in the constructed α-MHC-eGFP promoter vector transduced bonemarrow mesenchymal stem cells which were induced cardiac differentiation. Itsuggested that the constructed α-MHC-eGFP promoter vector was able to trace thecardiac differentiation;3) The bone marrow mesenchymal stem cells which weremodified in extracellular surface with50and250μg/mL MI1respectively wereadhesion and proliferation in in vitro culture assay, however, most of these cellsexperienced cell death after72h. While, the cells which were modified with large than250μg/mL MI1were fail to adhesion and most of the cells experienced cell death. Invivo studied showed that some of MI1modified bone marrow derived mesenchymalstem cells were able to target in infarcted myocytes via vein injedtion.Conclusions：1) The constructed of thymosin β4vectors were able to be expression ineukaryotic cells;2) the constructed α-MHC-eGFP promoter vector was able to tracethe cardiac differentiation of stem cell;3) The suitable concentrations of MI1for surface modification of bone marrow mesenchymal stem cells was around50μg/mL.