| Objective(1)Eukaryotic expression vector of hypoxia-responsive vascular endothelial growth factor(vascular endothelial growth factor,(VEGF)was constructed.and establish internal and external delivery methods and evaluate their effectiveness.(2)Using polylactic acid-glycolic acid copolymer(Polylactic acid-glycolic acid copolymer,PLGA)as drug carrier,to investigate the effect of carrier size on the release of different drugs,such as chemical drugs,nucleic acids,and so on.The particle size parameters of different drug carriers were optimized.(3)According to the characteristics of acute oxidative stress and long-term hypoxia microenvironment after myocardial infarction,a modular and controllable layer-by-layer assembly technique was used to develop an intelligent drug delivery system with acute antioxidant and hypoxic response to VEGF regulation.(4)In vitro cell models and myocardial infarction mouse models,the myocardial protection and myocardial infarction repair function of the above intelligent drug delivery system were systematically studied,in order to establish a new intervention method of myocardial infarction.Methods(1)Using gene recombination technology,on the basis of eukaryotic expression vector p GL4.73 [h Rluc/SV40](p SV),two tandem hypoxia sensitive EPO enhancers were inserted into the upstream of promoter SV40 to construct a hypoxia responsive expression system(p EPO-SV).Sea-kidney luciferase(Rluc)was used as downstream reporter gene to evaluate its hypoxia sensitivity.Then,VEGF165 gene was inserted into p EPO-SV plasmid instead of Rluc,and p SV plasmid without EPO enhancer was inserted into p SV plasmid as control.p EPO-SV-VEGF and p SV-VEGF expression vectors were obtained respectively.In vitro,p SV and p EPO-SV plasmids expressing Rluc or VEGF165 were transfected into 293 T cells,respectively.After treatment with normoxic and hypoxia for different time,the expression of Rluc or VEGF165 was used to identify the hypoxia-induced function of the constructed vector.(2)Two kinds of PLGA particles,nanometer and micron,were prepared by phacoemulsification and mechanical stirring emulsification,respectively.In addition,PLGA nanoparticles were used for plasmid delivery and functional verification in 293 T cells in vitro,which laid the foundation for drug carrier selection for different purposes.(3)The PLGA nanoparticles were prepared by phacoemulsification.The PLGA nanoparticles contained melatonin,an antioxidant drug,and then the charge interaction theory was used to prepare the nanoparticles,which contained the antioxidant melatonin,and the core of the nanoparticles was PLGA nanoparticles.Multilayer nano-drug systems with "protective layer @hypoxia-sensitive VEGF system @ antioxidation nucleus" structure were prepared by layer-by-layer accumulation method.The physicochemical properties were characterized by transmission electron microscope(TEM),)laser particle size and potential analyzer,Fourier transform near infrared spectroscopy(FTIR),and the structure and function of the plasmid were characterized by circular dichroism spectrometer and animal in vivo imaging instrument.The sustained release curves of plasmid and melatonin were determined.(4)Primary cardiomyocytes were extracted from the heart of newborn SD rats and divided into the following five groups: 1.blank untreated groups,2.oxygen-serum deprivation group,3.oxygen-serum deprivation melatonin nanoparticles treatment group,4.oxygen-serum deprivation melatonin / p SV-VEGF plasmid nanoparticles treatment group,5.oxygen-serum deprivation melatonin / p EPO-SV-VEGF plasmid nanoparticles treatment group.Apoptosis was detected by Tunel staining,antioxidant activity was detected by reactive oxygen species detection kit,and RNA expression of the target gene VEGF involved in plasmid treatment group was detected by RT-PCR.On the basis of this,the model of myocardial infarction in mice was established by ligating the left anterior descending branch of coronary artery,and was randomly divided into the following groups: 1.blank myocardial infarction group,2.myocardial infarction melatonin nanoparticles treatment group,3.myocardial infarction p EPO-SV-VEGF plasmid nanoparticles group,4.myocardial infarction melatonin-p EPO-SV-VEGF plasmid nanoparticles treatment group.The effects of Tunel staining on the early apoptosis of the model of myocardial infarction in mice were evaluated,and the cardiac function of the mice was detected by echocardiography.Massion staining and immunohistochemical staining were used to evaluate the infarct size and vascular density of the model of myocardial infarction in mice to verify the therapeutic efficacy of the system in vivo.Results(1)In the construction of plasmid recombination,the successful insertion and correctness of EPO enhancer and VEGF165 gene were confirmed by restriction enzyme digestion,PCR and DNA sequencing.There was no significant difference in the expression of reporter gene Rluc and target gene VEGF165 in 293 T cells induced by hypoxia,but the expression of Rluc and VEGF165 in recombinant p EPO-SV was significantly higher than that in p SV,.The expression of reporter gene Rluc and target gene VEGF165 in hypoxia-induced p EPO-SV plasmid and proto-plasmid p EPO-SV were significantly higher than those in p SV,under normal culture conditions.The results showed that the constructed p EPO-SV plasmid had typical hypoxia-induced expression activity.(2)The size of the nano-particles was mainly distributed in 200-300 nm,and the diameter of micron particles was mainly in 2-4?m;The carrying capacity of melatonin in PLGA nanoparticles was14.3% and 14.1%,respectively.In the field of drug release,there is a significant early release phenomenon of nano-particles,and the release time of micron particles is longer,the sustained release time can be up to one week or so.In the aspect of nucleic acid sustained release,the sustained release ability of nano-particle carrier is better than that of micro-particle carrier.As for the ability to carry drugs into cells,PLGA nanoparticles with relatively small particle size were easier to enter or bind to cells,and reached the maximum value at 12 hours after incubation,while the micron particles were relatively slow,and the maximum value appeared 24 hours after co-incubation.After delivery of p EPO-SV and p SV by PLGA nanoparticles in 293 T cells in vitro,the detection of reporter gene Rluc under normal and hypoxic conditions showed the same results as that of the first part.The results showed that PLGA nanoparticles could be used as effective carrier for plasmid delivery.(3)The results of TEM and SEM showed that the structure of the prepared nanoparticles was regular,uniform in size and a typical spherical structure.At the same time,the results of TEM,laser particle size analyzer and potential analyzer show that the particle size of the structure increases gradually with the layer-by-layer superposition effect,and the positive and negative interactions can also be observed in each layer.The results of CD showed that the plasmid could maintain the stability of the structure in the multilayer structure,and the in vivo imager showed that the multilayer structure could be used as an effective carrier for intracellular delivery of plasmacytes.The slow release of melatonin in multilayer particles was almost complete within 48 hours compared with that of pure nanoparticles.The slow release of plasmid was relatively slow and lasted for a week.(4)Compared with the untreated hypoxic serum deprivation group,melatonin-carrying nanoparticles significantly reduced the apoptosis of cardiomyocytes in hypoxia-serum deprivation environment(P < 0.01),and the level of reactive oxygen species(Ros)was also significantly inhibited(P < 0.01).The results of PCR showed that compared with the nanoparticles treated with p SV-VEGF plasmid,the expression of target gene VEGF in the group treated with p EPO-SV-VEGF plasmid was significantly higher than that in the control group(P < 0 05).The results of in vivo myocardial infarction: compared with the untreated control group,the results of Tunel staining showed that the treatment group with melatonin nanoparticles significantly reduced the apoptosis of cells in the early stage of myocardial infarction model and improved the cardiac function(P < 0.05).The vascularization of myocardial infarction area in the treatment group with p EPO-SV-VEGF plasmid was significantly increased.At the same time,melatonin and p EPO-SV-VEGF plasmid nanoparticles in the treatment group showed synergistic effect of myocardial infarction repair,ultrasound,The results of Massion staining showed that the treatment group of melatonin and p EPO-SV-VEGF plasmid nanoparticles had the best effect on the repair of myocardial infarction(P < 0 05).Conclusion(1)A typical eukaryotic expression system of hypoxia-responsive VEGF has been successfully established.This expression vector may have an important application prospect in tissue damage diseases such as ischemia and hypoxia.(2)As a chemical drug carrier,PLGA nanoparticles are more suitable for acute tissue or cell protection,and micron particles are more suitable for chronic sustained protection.As a carrier of nucleic acid plasmid,PLGA nanoparticles is a better choice than micron particles,and an effective delivery method of intracellular plasmids has been established.(3)The bifunctional nano-carriers carrying melatonin and hypoxia-sensitive VEGF plasmid were successfully prepared by layer-by-layer accumulation method.The co-delivery of VEGF plasmid and melatonin by this vector demonstrated that the system has a significant protective effect on myocardial cells under hypoxia-serum deprivation model in vitro,and can play a significant synergistic effect in the repair of myocardial infarction,promote the repair of myocardial infarction. |
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