| Background:Some research indicated that stem cells could not only replace the necrosis cardiomyocytes and increase the number of functional cardiomyocytes , but also raise the density of vascellum in the post infarction scars and improve the cardiac function. These results observed before would introduce a new therapeutic strategy to the field of cardiovascular diseases. It has been proved in several studys in vitro or in vivo that autologous bone marrow stem cells could rescue ischaemic myocardium,induce neovascularization and preserve left ventricular function. In this study we transfected HRE-VEGF into bone marrow stem cells, in order to make clear whether BMSCs transfected with HRE-VEGF could protecte cardiomyocytes,induce neovascularization,prevent VEGF overexpression and greatly improve the cardiac fuction after ischemic and hypoxia。Part I: Separation and cultivation of rats bone marrow mesenchymal stem cells and establishment of animal modelSection one: Separation and cultivation of rats bone marrow mesenchymal stem cellsObjective:To build a method to cultivate rat MSCs in vitro, provide the cell resource for autologous bone marrow stem cells transplantation.Methods: After rat MSCs have been separated and depurated, the cell cycle were detected by flow cytometryResults: when MSCs were cultivated in vitro for 10～14 days, they confluensed and 75 percent of these cells were in the phase of G0/G1.Conclusion: rat MSCs could be separated and amplified through the adhesiveness character in vitro grew and amplified, these cells could be used in cell transplantation.Section two: Establishment Of Animal ModelObjective: to establish rat myocardial infarction model with liquid nitrogen frozenMethods: to establish myocardial infarction animal model with the method that anterior wall of left ventricle of rat was contacted directly by metal stick soaked in liquid nitrogen.Results: cordis hypersound data showed that there was significant difference between heart function before modeling and after modeling, the heart function before modeling was better than that after modeling.Conclusion: this kind of myocardial infarction animal model is reliable because of the fixed location.Part II: Construction of HRE-VEGF-Expression VectorSection one: The Construction of VEGF-Expression VectorObjective: Clone the cDNA of VEGF, combine cDNA with the expression vector to get the HRE-VEGF -Expression VectorMethods: According to CDS domain of VEGF published in genebank we amplified the HRE-VEGF gene with RT-PCR from Hep G2 cells and clone the gene to pCDH1-MCS1-EF1- copGFP, then we transfected VEGF- expression vector into DH5αcompetence bacterium, The masculine clone bacterium were choosed, double enzyme-cutting and sequencing were conducted to verify the cDNAResults: double enzyme cutting and sequencing indicated the cDNA we got were the same sequenc to CDS domain of VEGFConclusion: Using RT-PCR could successfully construct VEGF-expression vectorSection two: The Construction of HRE-VEGF Expression VectorObjective: To clone the hypoxia response element tandem repetitive sequence, and to insert it into the objective gene expression vector which was proved by sequencingMethods: complementary single strand DNA on head and end section was designed artificial by the end of erythropoietin of human being, and 9HRE was gotten by enzyme-cutting 3 times and connecting. Then double strands DNA of HRE with enzyme-cutting site and 9 cascade connections was gained by renaturation and elongation. The double strands DNA was sequenced after cloned to the expression vector.Result: both the results of double enzyme-cutting verification and sequencing indicated that 9HRE-VEGF which was proved by sequencing matched to the designed one.Conclusion: the method of isocaudarner can construct 9 copy hypoxia response element and 9HRE-VEGF expression vector successfully.Part III: lentivirus mediated HRE-VEGF gene transfection of MSCs and the regulation effect of HRE on the transgenic stem cells in hypoxia and oxygen recovery myocardial cellsSection one: HRE-VEGF gene transfection bone marrow stem cells which was mediated by lentivirusObjective: to transfect stem cells with HRE-VEGF gene, and to establish gene transfected bone marrow stem cells with lentivirus, in order to study the regulation effect of HRE to the cells with transduction VEGF gene in hypoxia and oxygen recovery myocardial cells.Methods: after amplification of 293T cells which were transfected by shuttle plasmid PCDH1 combined with HRE-VEGF gene, gene recombination was carried out by the system of lentivirus transfection composed by 3 plasmids . Bone marrow stem cells of rat were then transfected by that recombination gene as trager and were detected its fluorescent expression.Results: On 24 hours and 48 hours after transfection, the strong fluorescent expression can also be detected in 293 T cells and MSCs.Conclusion: Lentivirus trager system with HRE-VEGF gene has high transfection efficiency in transfecting MSCs of rat.Section two: Effects of Transgenic Stem Cells On Hypoxia CardiomyocytesObjective: to study the regulation of hypoxia response element to the gene expression of hypoxia and oxygen-recovery myocardial cells transfected VEGF gene of human being.Motheds: to primary culture myocardial cells, and divided them into different groups with the type of stem cells and the time of hypoxia and oxygen recovery. After being fixed, the cells in each group were stained with immunofluorescence to detect the expression of VEGF proteinum; RT-PCR was used to detect the content of VEGF-mRNA of human being; content and type of VEGF was detect by ELISA from cell culture fluid.Results: VEGF- mRNA and VEGF were detected in hypoxia group, and VEGF- mRNA and VEGF were found in the group without hypoxia.Conclusion: the regulation effect of HRE to the transfected VEGF gene has been proved, during MSCs taking along HRE-VEGF gene were added into rat myocardial cells isolated cultured during the process of hypoxia and oxygen recovery.Part IV: Gene-modified bone marrow stem cells autografting therapy in rat ischemic cardiomyopathy modelObjective: to investigate if MSCs transfected HRE-VEGF gene by autografting has more significant effect than MSCs without transfection in improving the heart function of ischemic cardiomyopathy, and to approach the therapeutic possibility about MSCs and gene used to treat myocardial ischemia.Methods: male Fischer 344 rats with weight from 200g to 250g were put into research, MI scar were made by liquid nitrogen frozen, cardiac function were observed by hemodynamics monitoring. These rats were divided into four groups: control group(A groups, n=20),DMEM group(B group, n=20),MSCs group (C group, n=20),transgene group (D group, n=20).Bone marrow were taken from rats'femur. adherence screening technique were adopted to separate,purify and amplify MSCs in vitro. Then MSCs were transfected by HRE-VEGF.Autologous bone marrow stem cells labeled by BrdU were injected into MI scar. After 4 weeks, cardiac function was monitored again in order to evaluate the heart function and scar infusion. Histology research were done to know the distribution of MSCs, otherwise, Changes in hematoplasma concentration of VEGF between post MI and posttransplant were got through the methods of ELISA. Cx-43,VEGF were also detected by ELISA to knowledge the effect of MSCs on neovascularization.Results: Rats'MSCs were transplanted into MI scar, after 4 weeks, data from hemodynamics monitoring indicated that there is no obviously different in LVSP between experiment groups and control groups. In C,D group, LVEDP were lower, the±dp/dtmax were more better than that of A,B group. In D group and C group, +dp/dtmax in D group were higher that of C group. MSCs transplantion could protect scar from being thin and dilate, moreover, cardiac function and scar infusion were more improved compared with A,B group and pretransplante. The improvement of cardiac function were more successful in D group (to C group , P<0.05).The capillary density in C,D group increased greatly in MI scar compared with A,B group(P<0.05),and the increasing of D group were more obvious than C group. Concentration of VEGF also increased after MI and came up to peak 1 week later, after 1 week, the data showed the decrease in concentration of VEGF.Conclusion: MSCs were observed still survived in the host myocardium, protected MI scar from thinning, inhibited the dysfunction of cardiac and improved the left ventricle function; these changes might attribute to the myocardium regenerate,neovascularization,extracellular matrix hyperplasy;Some cytokines might play very important role on these changes. Expression of VEGF could also release the cell apoptosis, reinforce neovascularization and improve the cardiac function.Transplantation of autologous bone marrow stem cells combined with HRE-VEGF was an effective treatment to ischemia myocardial disease... |
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