An Experimental Study Of UTMD Combined With Nanoparticles To Mediate TGF?1 And TIMP1 To Stabilize Atherosclerotic Vulnerable Plaques

Atherosclerosis is a chronic disease caused by vascular intima of endothelial injury and inflammatory reaction.The plaque rupture after shedding emboli and subsequent thrombosis is the main cause of acute cardiovascular and cerebrovascular ischemic stroke,and plaque rupture occurs almost all on the basis of vulnerable plaque.Therefore,the stability of atherosclerotic plaques is the prerequisite and basis to prevent the occurrence of atherosclerosis and its complications.In vulnerable plaque,the integrity of the fibrous cap and dynamic balance of rupture resistance mainly depends on the synthesis and degradation of extracellular matrix.Matrix metalloproteinase and tissue inhibitor of metalloproteinase(tissue inhibitor of metalloproteinase,TIMP)plays an important role in maintaining the balance.TIMP1 is mainly associated with atherosclerosis.Transforming growth factor beta 1(transforming growth factor Betal,TGF?1)can promote the synthesis and secretion of extracellular matrix,content and activity regulation of MMPs,stimulate synthesis of TIMP,as the key to keep between plaque inflammation and fibrosis of atherosclerotic balance.TGF?1 is a protective factor for atherosclerosis originating and at the same time is also a very important factor in maintaining the stability of plaque.This experiment used iron oxide nanoparticles with TGF?1 and TIMP1 two kinds of plasmids which were wrapped in ultrasound targeted microbubble destruction(UTMD).The loaded objective plasmids were successfully transferred to internal atherosclerosis plaques,reaching the extracellular matrix,blocking the degradation of extracellular matrix,promoting the synthesis of extracellular matrix.This method can stabilize the vulnerable plaques for therapeutic purposes.This study was mainly divided into three parts:Part ? construction and function verification of special plasmidObjective:to construct the expression vector targeting macrophage pSNAV2.0-TGF?1-TIMP1 recombinant adenovirus plasmid,and to verify the objective function,to prepare materials for post transfection experiments.Methods:amplified from primer synthesis to target gene,to construct recombinant adenovirus expression vector and sequencing.Cell culture,plasmid extraction and cell transfection were carried on.Inverted fluorescence microscope was used to observe the expression of recombinant adenovirus which was transfected into HEK293 cells.qRT-PCR technology and Western-Blot technology were used to detect the expression of TGF?1 and TIMP1.Results:qRT-PCR showed that the expression levels of TGF?1 was 7.17%±0.15%,it was significantly higher than the control group(2.62%±0.47%)and the vector group(2.38%±0.33%);the expression content of TIMP1 was 4.84%±0.15%,which was significantly higher than the control group(1.98%±0.06%)and the vector group(2.27%±0.08%).While Western-Blot showed that CT value in overexpression of TGF?1 group was 11946.18,it was significantly higher than the control group(3882.841)and the vector group(4488.205);CT value in TIMP1 overexpression group was 15241.34,which was significantly higher than the control group(5610.619)and vector group(5579.69).The plasmids were successfully constructed.Conclusion:the special plasmid was successfully constructed and can be used for post transfection experiments.Part ? experimental study on promoting TGF?1-TIMP1transfected macrophages by using UTMD nanoparticlesObjective:UTMD was combined with iron oxide nanoparticles for gene transfection in vitro,the effect of UTMD gene transfection combined with nanoparticles in cells was assessed by detecting the transfection efficiency and gene therapy.Methods:iron oxide nanoparticles targeting macrophages were prepared,cells were divided into 3 groups:A group for control group;B group for plasmid+nanoparticles group;C group was UTMD+plasmid+nanoparticles group.Gene transfection experiment was begun after choosing the best ultrasonic irradiation conditions.The transfection rate and cell apoptosis rate were detected by flow cytometry after transfection 48 hours,the effect of gene therapy were detected by Western-blot and real time-PCR technology;cell activity was detected by CCK-8 cytotoxicity test.Results:UTMD combined with iron oxide nanoparticles can promote plasmid transfection,transfection of the experimental group was the highest,about 1.8 times as much as the plasmid+nanoparticles group;the apoptosis rate of the experiment group was the highest,about 8 times as much as the plasmid+nanoparticles group.PCR showed that the expression of TGF?1 was 26.77%±3.67%,which was significantly higher than the control group(9.58%±0.75%)and the vector group(8.75%±0.63%);the expression of TIMP1 was 19.82%±1.23%,which was significantly higher than the control group(6.23%±1.01%)and the vector group(6.28%±0.59%).Western-Blot showed that CT value of overexpression of TGF?1 group was 19944.794,which was significantly higher than the control group(2722.548)and the vector group(2811.669);while CT value of TIMP1 group was 22625.167,which was significantly higher than the control group(5116.447)and the vector group(5955.539).CCK8 test showed that the cells in the control group had the highest survival rate(0.977±0.005),while in experimental group was lowest(0.976±0.004),there was no significant difference between groups.There are no obvious effect on the UTMD activity of nanoparticles on cells.Conclusion:UTMD combined with nanoparticles was a safe technology.It can promote TGF?1-TIMP1 transfected macrophages and induce apoptosis in macrophages,inhibiting the growth of vulnerable plaques.This method can provide the experimental basis for the gene therapy of atherosclerotic vulnerable plaques.Part ? experimental study on promoting TGF?1-TIMP1 transfected atherosclerotic vulnerable plaques by using UTMD nanoparticlesObjective:to evaluate the changes of molecular detection of target gene by the combination of UTMD and nanoparticles mediated plasmid transfection of rabbit aorta plaques,and to evaluate gene transfection efficiency in vivo through the abdominal aorta at the level of plaques ultrastructure,and to evaluate the feasibility of gene transfection in vivo.Methods:to establish atherosclerotic rabbit model of vulnerable plaques,experimental groups:group A:normal group;group B:model group;group C:the plasmid+nanoparticles group;group D:UTMD+plasmid+nanoparticles group;group E:UTMD+plasmid+nanoparticles+magnetic field group.It was assessed by conventional ultrasound and contrast enhanced ultrasound before and 10 weeks after the experimental treatment of abdominal aortic plaques.And gene therapy was detected by Western-Blot technology.HE staining of vulnerable atherosclerotic plaques in rabbit abdominal aorta was used to measure and analysis average intima thickness(IT),average media thickness(MT),plaque thickness(PT),intima-media thickness ratio(IT/MT)and plaque-intima thickness ratio(PT/IT)for each slice in the abdominal aorta of rabbit.Results:10 weeks after operation,the echo of intima and media of abdominal aortic were enhanced in the experimental groups by using two-dimensional high frequency ultrasonography.B group was the most obvious,the increase of intima-media thickness in group C,group D and group E was less than group A and group B.The vulnerable plaques increased obviously in the group B.but the growth rate in group C and group D decreased compared with B group.E group of vulnerable plaques didn't increase obviously.PSV1 at abdominal aorta vulnerable plaques and its upstream of PSV2 in 10 weeks after operation were no significant difference among three groups.Contrast enhanced ultrasound showed that contrast agent inside the vessel was transported to the vessel wall after the external magnetic field was given.Conclusion:the combination of UTMD and nanoparticles promoted targeted transfection of recombinant plasmid and significantly induced apoptosis in macrophages in atherosclerotic vulnerable plaques,and effectively inhibitted the growth of vulnerable plaques.This method can effectively promote the gene transfection in vivo.