Construction And In Vitro Study Of Mechanical Heart Valve Coated With TPA And VEGF165 Genes By Electrospinning

Part one:Construction of eukaryotic expression vectors including human tPA and VEGF165 genesPurposes:Obtaining genes of human tissue-type plasminogen activator (tPA) and vascular endothelial cell growth factor 165 (VEGF165) from template by gene clone, and inserting them into the two multiple clone sites of eukaryotic expression vector, plasmid pIRES, to construct two plasmids vector: pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 which contain the two target genes.Material and methods:Primers containing restriction enzyme cutting sites of endonuclease, EcoR I and Xba I, were designed according to completely coding sequences of human tissue-type plasminogen activator gene (NM₀00930.3) and vascular endothelial cell growth factor gene (NM₀01025368). Polymerase chain reactions were applied to amplified the target genes, using plasmid pcDNA3.1-Myc-His B(-)/ tPA and cDNA library as templates. The target genes were obtained from gelatins after agarose gel electrophoresis subsequently. Linearization of plasmid pIRES was made by EcoR I and Xba I, and then tPA gene and VEGF165 gene were inserted into the two multiple clone sites of the vector respectively. Recombinant eukaryotic expression vectors, pIRES-VEGF165-tPA and pIRES-tPA-VEGF165, were obtained from different inserting orders. Competent cell DH5 a was transformed by recombinant vectors. Polymerase chain reactions of positive bacterial colony were performed to detect the target genes in the plasmids. DNA sequencing was used to affirm the vectors in the end.Results:Two pieces of target gene, sized about 1721bp and 1151bp, were observed from agarose gel electrophoresis, which fit the sequence of human tPA and VEGF165 genes. Positive plasmids could be found from polymerase chain reactions of bacterial colonies after transformation by recombinant vectors pIRES-VEGF165-tPA and pIRES-tPA-VEGF165. The result of DNA sequencing showed the target genes were inserted into the vector correctly except several synonymous mutation, such as CTG to CTA in tPA gene, which will not affect the expression of the proteins.Conclusion:Recombinant eukaryotic expression vectors pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 including genes of human vascular endothelial growth factor 165 (VEGF165) and tissue-type plasminogen activator (tPA) were constructed successfully.Part two:Transfection research of eukaryotic expression vectors including human tPA and VEGF165 genes in vitroPurposes:To verify the instantaneous transfection efficiency of the eukaryotic expression vectors pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 in the human umbilical vein endothelial cells, test the transcription and translation of the target genes, and the secretion and function of the proteins, while find out the differences between the two plasmids in these fields in vitro.Material and methods:Plasmid pGFP, which contain the gene of green fluorescent protein, and the eukaryotic expression vector pIRES-VEGF165-tPA or pIRES-tPA-VEGF165 were transfected into the human umbilical vein endothelial cells (EA.hy926) at the amount rate of 1:1 by Attractene Transfection Reagent kit. The instantaneous transfection efficiency 12 hours,24 hours, and 48 hours after transfection were confirmed by counting the positive cells giving out green fluorescence, making the plasmid pIRES and PBS solution as the negative controls. Quantitative real-time RT-PCR and Western blot were used respectively to measure the content of mRNA and proteins of tPA and VEGF165 in the cells after transfection. Also, the supernatant of cells were harvested to check out the concentration of secretory proteins of tPA and VEGF165 in it by ELISA test. The function of the proteins were evaluated by MTT test and tPA activity assay kit.Results:By counting the positive cells giving out green fluorescence, the instantaneous transfection efficiency of pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 in the human umbilical vein endothelial cells (EA.hy926) were found reached the top 24h after transfection, about (15.6±3.1)% and (16.3±2.9)%, and decreased subsequently. There was no significant difference in the transfection efficiency between the two vectors (P>0.05). The results of quantitative real-time RT-PCR and Western blot showed that the levels of mRNA and proteins of tPA and VEGF165 in the EA.hy926 cells transfected with pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 were higher than the pIRES and PBS groups, with a significant difference (P<0.05). The levels of mRNA and protein of tPA in the pIRES-tPA-VEGF165 was higher than that in pIRES-VEGF165-tPA group, with a significant difference (P<0.05), but there was no significant difference in levels of mRNA and protein of VEGF165 between the two groups (P>0.05). And the ELISA, MTT test and the tPA activity assay approved that the content of tPA and VEGF165 protein in the supernatant of cells transfected with pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 were much more than that in the pIRES and PBS groups, with a significant difference (P<0.05), but there was no significant difference in the content of the two protein between pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 group (P>0.05) Conclusion:The eukaryotic expression vectors pIRES-VEGF165-tPA and pIRES-tPA-VEGF165 can transfect into in the human umbilical vein endothelial cells, and the positive cells can express and secrete functional tPA and VEGF165 proteins. The content of mRNA and protein of tPA in the pIRES-tPA-VEGF165 group was higher than that in the pIRES-VEGF165-tPA group, but it does not mean there is significant difference in the function of pIRES-VEGF165-tPA and pIRES-tPA-VEGF165.Part three:Preparation of compound terylene coated with cross-linking gelatin microspheres by electrospinning and the research of its physical and chemical charactersPurposes:For preparing a new compound material, which can release plasmid chronically, with cross-linking gelatin microspheres as the controlled release medium and terylene by electrospinning, and evaluating physical and chemical characters of it, such an the stability of its structure, the cellular compatibility, and the drug loading ability, looking forward to use it in the build of a new mechanical heard valve.Material and methods:The electrospinning solution was made of cross-linking gelatin microspheres and 10% polyvinyl alcohol (PVA) solution at different W/V ratio. A piece of terylene was put on the collector to accept the fiber produced by electrospinning. The process of electrospinning was performed at proper parameters, such as voltage and distance from nozzle to collector, to produce a compound terylene coated with ultrafine fibers. The surface structure of this material was observed by scanning electron microscope (SEM). The stability of its structure was tested by shaking the material and distilled water in a shake with thermostatic shaker. MTT test was used to estimate the effect of the compound terylene to human umbilical vein endothelial cells (EA.hy926). The pIRES-tPA-VEGF165 plasmid and the compound material were mixed at different mass ratio, the drug loading ability of the material was confirmed by detecting the residual content of plasmid in the solution, making single terylene and single fiber as control groups. The compound material with plasmid was put in PBS, and shaked at 37℃and 30rpm. Samples of the solution were collected every 48h. A line graph of the drug-released was plotted by the absorbance at 260nm.Results:When the W/V ratio of cross-linking gelatin microspheres and 10% polyvinyl alcohol (PVA) solution was less than 1/100, a homogeneous ultrafine compound fibrous material can by obtained by electrospinning at room temperature,16-20kV of voltage,10-12cm of collecting distance, and 2-4ml/h of infusing velocity. Cross-linking gelatin microspheres were spread among the fibers by scanning electron microscope, and its density increased as more microspheres were put into the solution. The structure of this material was intact after shook at 30-200rpm, and no defluvium was observed on the surface. MTT test approved that there was no significant difference in the effect to the multiplication of EA.hy926 cells among compound terylene, single terylene and single fiber (P>0.05). The drug loading ratio of the material reached the top when the mass ratio of the pIRES-tPA-VEGF165 plasmid and the compound material was 1:100, and could not be improve by increased content of plasmid. The line graph of the drug-released showed that plasmid could be released gradually from the material, the releasing speed was faster in the first two days, became slow from 8th day, and the total content of the plasmid decreased from 12th day.Conclusion:Cross-linking gelatin microspheres including plasmid could be combined with terylene by electrospinning. The compound material can keep structure integrality under shearing force, had perfect compatibility with human umbilical vein endothelial cells, could bring and release plasmid gradually in vitro, so it might be used in the build of a new mechanical heard valve.Part four:Transfection assay of compound terylene coated with cross-linking gelatin microspheres including tPA and VEGF165 genes in vitroPurposes:To evaluate the transfection efficiency of compound terylene coated with cross-linking gelatin microspheres including tPA and VEGF165 genes and the secretion of target proteins in human umbilical vein endothelial cells. Material and methods:The compound terylene coated with cross-linking gelatin microspheres including pIRES-tPA-VEGF165 and pGFP, mixed at mass ratio of 1:1, was put into the complete cell-culture medium. The supernatant of the solution was collected each day of the first six, respectively. Sono Vue, a ultrasound microbubble contrast agent (UAMBCA), was added into the supernatant to transfect the EA.hy926 cells, assisted with ultrasonic irradiating at 1 MHz,2 W/cm2 for 60 sec. The instantaneous transfection efficiency were confirmed by counting the positive cells giving out green fluorescence 24h later. The compound material including only pIRES-tPA-VEGF165 was use to transfect the EA.hy926 cells in the same way, making plasmid pIRES and PBS as the control groups. ELISA were performed to detect the content of tPA and VEGF165 proteins in the supernatant 24h after transfection.Results:Positive cells were found in group transfected with pIRES-tPA-VEGF165/pGFP on each day of the six by fluorescence microscope. The transfection efficiency was higher in the first two days, about 20%, and then decreased. No positive cells was seen in the control groups. The result of ELISA showed that tPA and VEGF165 proteins were detected in the supernatant of pIRES-tPA-VEGF165 group every day, whose tendency was consistent with the transfection efficiency. And the content of them were more than those in control groups, with a significant difference (P>0.05). A little tPA protein was found in the supernatant of pIRES and PBS groups, without a significant difference between them (P<0.05),but no VEGF165 protein.Conclusion:Assisted with ultrasonic irradiating and ultrasound microbubble contrast agent, compound terylene coated with cross-linking gelatin microspheres including target genes could transfect human umbilical vein endothelial cells in vitro, which secreted homologous target proteins.