The Practical Study Of Target Gene Carried By Targeting Constrast Medium Which Stabilize Atherosclerotic Plaque

As we know,atherosclerotic cardiovascular disease(ASCVD) is called"number one killer"in the developed country.The disease incidence of ASCVD is also growing higher and higher in the developing country.WHO predict that to 2020, the ASCVD will become the first death cause in the world when the case-fatality rate of infectious disease is effectively controlled in the world extent. The ASCVD is one of the large group of disease which hazard the human health seriously,the rate of its causing death and disability is high, the precaution,diagnose and treat for the ASCVD are always the investment emphasis of every country government.But our key issue at present is to discriminate the instability plaque ,carry out clinical intervention and enhance stability of the plaque. Gene therapy have a good future to stabilize atheromatous plaque.We heightened the level of HDL significantly in rat blood plasm by over-expressing the receptor gene of HDL,which influence the occurrence and proceeding of atherosclerotic plaque.,diminish the exterior factor that induce plaque rupture.The key point and foundation of gene therapy is precise transportion into the target organ or cell. As a magnetic nanometer wearing-medicine system , USPIO can be used to transport theoretical gene,the purpose of our experiment is to seek a therapy which have high diversion efficiency, lesser adverse effect and convenient application through specific englobement of macrophage and the direct effect of apposition magnetic field.Method1.Gene amplificationWe get pcDNA array of Apo-A-I and SR-BI from the Genebank,then design the primer according to the needs of experiment. We extract the Apo-A-I,SR-BI mRNA from the hepatic tissue of rat after homogenate, segregation, precipitation, dissolvation, According to the method of TAKARA One–step RT-PCR,we amplificate Apo-A-I gene and SR-BI recombination gene through the primer.we connect the production of PCR and the carrier of pGEM-T, transform the connective into the Bacterium coli TG ,insert the Apo-A-I and SR-BI gene into the expression vector after screening the positive clone and shearing the intermedial cloning vector—pGEM-T/Apo-A-I , pGEM-T/SR-BI and expression vector—pcDNA3.1(+) by double-enzym.Then we get recombinated expressing vector—pcDNA3.1(+)/SR-BI and pcDNA3.1(+)/Apo-A-I.After the appreciation by enzyme shearing,we observate the expression of gene through transient transfection hepatic cell line of rats by eukaryotic expression plasmid.2.The preparation of bangosomeWe preparate the magnetic bangosome with positive electricity by antiphase evaporation method ,let the bangosome and DNA form compounds by electrostatic interaction;then we observate the appearance and give the single bangosome spectrum analysis.We measure the particle diameter distribution and the mean diameter by Zetasize laser diffraction grainsize analyzer; measure the rate of DNA envelopment after high speed centrifugation;then we observate the responsibility of the compounds in the vitro magnetic field.3. The establishment of ATHSC animal model50 Male Wistar rats which is divided in two group ,each rat's body mass is about 200-220g. The 4 rats of control group are bred with the elementary bait vessel, the 46 rats of experimental group are bred with hypsifat bait vessel,which are 3% cholesterin, 0.5% sodium cholate, 0.2% propylthiouracil, 5% plantation white sugar, 8% leaf fat and 83.3% elementary bait vessel.Each of the rats in two groups is fed with 20g bait vessel, and allowed to hydroposia freely. The experimental group is peritoneal injected with vitamin D60万IU/kg adunam vicem when the feeding begin. The control group is peritoneal injected with the isovolumic normal saline.The two groups are surveyed the 4 items of blood-fat when time is the feeding begining,the third week and the 6th week.When the 6th week is coming,4 rats in each group is killed, their abdominal aortas(AO) are dissociated and took out,One part of the AO is used for gross sample,the other part of the AO is used for transection pathological section.We use different pathological staining to identify different reconstituent in the plaque. The gross samples are stained by oil red.We use projection panel counting method to calculate the common area of every aortic tunica intima,area of aorta and abdominal pectoralis,plaque area which is positive stained by oil red O,then we get the percentage of plaque area.We also should count the number of sections which have the plaque or not.At last,we useχ2 analyses though the disposal of SPSS11.5 English software.At the same time, the reconstituent of transection pathological section and the stegnotic extent of lumina are analyzed.4. Gene therapyThe 42 rats which has been built up to artery atheromatous plaque models are divided into four groups completely in random.First group(A Group),12 rats are injected magnetic bangosome/DNA compounds containing Apo-A-I gene from their vena caudalis.The second group(B group):12 rats are injected magnetic bangosome/DNA compounds containing SR-BI gene;The third group(C group):12 rats are injected the two magnetic bangosome/DNA compounds containing the 2 genes above-mentioned.The forth group(D group):As the blank,6 rats are injected magnetic bangosome /DNA compounds containing none of gene .The injection dose is 0.32ml(correspondent to 40ug genes)for each rat.After injection,each rat is bound to rubidium-iron-boron rare earth magnet(500mmT field strength) over its left kidney about 4 hours.When the gene therapy has done at the third and 6th week ,the rat were surveyed the 4 items of blood-fat and then they were killed,.we dissociate and take out their abdominal aortas(AO) which is used for gross sample and transection pathological section.Pathological and statistical method are as we've said above.Consequence1. Gene amplificationThe RNA of rat hepatic tissue is drew with TRIZOL agent,there are 3 straps we can see conspicuously which are 28S,18S,5S. The value of OD260/280B is 1.85 detected by ultraviolet spectrophotometer,which indicate that we've gotten good quality of RNA templat. The intermedial cloning vector—pGEM-T/ Apo-A-I and pGEM-T/SR-BI are identificated by double-enzyme Shearing.The size of objective gene fragment is about 780bp to 1602bp,just as the prediction. The alliance gene is also same as the prediction after sequencing. The recombinate expression vector of pcDNA3.1(+)/SR-BI and pcDNA3.1(+)/Apo-A-I are also identificated by double-enzyme Shearing,the size of fragement is as the prediction which showed us that we'd gotten good recombinate expression vector. The detection of flow cytometry and Western Blotting manifested that the gene of SR-BI,APO-A-I can express in Wistar hepatic cells perfectly.2. Preparation of the bangosomeThe particle diameter of themagnetic bangosome prepared with antiphase evaporation method distribute in the extent of 50-200nm, the particle diameter of magnetic bangosome /DNA compounds distribute in the extent of 200-400nm. The single of bangosome contains silicon, manganum, zinc, iron etc.The rate of envelopment is 81.4%—the highest when the rate of electric charge between DNA and bangosome is 1:7. The experiment of vitro magnetic responsiveness demonstrate the magnetic bangosome /DNA compounds has a good character of magnetic responsiveness.3. Establishment of ATHSC animal modelThe rat is haemospasiaed from the orbital vein at the time of beginning ,the third week and the 6th week after hypsifat feeding.We need to detect total cholesterol(TC), triglyceride ( TG ) ,high density lipoprotein ( HDL ) and density lipoprotein(LDL).Those biochemical data dealed by statistical treatment(P<0.01) indicate that blood fat is heightening constantly with the prolongation of hypsifat feeding.The artery of control group is soft and stretchable, their endomembrane is smooth. The atherosclerotic plaque occur in all of the experimental group,the vessel wall is stiff and thicken obviously, they appear to white barred eminentia.The gross pathologic stained by oil red O showed us that there are many irregular red eminentia on the surface of endangium; hyperplasia endothelialis is obviously observed with the microscope; a great quantity of foam cells aggregated under the fibrous membrane;the plaque is outstanding to the lumina.All of above are typical appearance of the plaque.4. Gene therapyThe atherosclerotic plaque is diminished in each gene therapy group, Part of the vessel walls become soft and recover flexibility;the endomembrane of lumina become smooth, the vessel walls are not thicken obviously. The gross pathologic stained by oil red O showed us that: the irregular red eminentia on the surface of endangium was diminished; the rate of lipid fringe area and fibrous plaque area is 9.17% and 1.11% in A1 group, 9.19% and 1.17% in A2 group; 9.23% and 1.13% in B1 group, 9.20% and 1.18% in B2 group; 9.21% and 1.13% in C1 group, 9.22% and 1.16% in C2 group.hyperplasia endothelialis is weakened under the microscope; there are manipulus foam cells aggregated under the fibrous membrane.In the control group, the vessel walls are still stiff and thicken obviously. there are many irregular red eminentia on the surface of endangium; the rate of lipid fringe area and fibrous plaque area is 32.25% and 1.66%,hyperplasia endothelialis is obviously under the microscope; a great quantity of foam cells aggregated under the fibrous membrane;the plaque is outstanding to the lumina.The consequence are typical appearance of the fragile plaque.The two random group comparison between the gene therapy and control group is analyzed withχ2 test by the English software of SPSS11.5 after we count the number of sections which have the plaque and not. The rate of plaque area is compared in the group treated with plaquegene therapy(3d week,6th week) and the control group,the disparation has statistical significance(P<0.01);The comparison between every gene therapy group treated 3 weeks and 6 weeks indicate no significant deviation(P>0.05). in 3 weeks, The comparison between the gene therapy group indicate no significant deviation(P>0.05). and in 6 weeks,the outcome is also no significant deviation(P>0.05).ConclusionOur findings manifest that: (1) It is effective and feasible that ultramicro- Superparamagnetic Iron Oxide (USPIO)can be used to transport theoretical gene as a magnetic nanometer wearing-medicine system. (2) The SR-BI,Apo-A-I gene can enhance the biological effect of HDL, accelerate the clearance of inflammation in the plaque and the LDL relative to MMPS after they reach the liver directly with the introduction of adventive magnetic field. Finally,they can raise the stability of plaque and play a role of gene therapy.