| Coronary heart disease(CHD)is gradually becoming one of the main diseases of threatened human health at the present stage.Although all kinds of treatments such as thrombolytic drug,interventional therapy and coronary artery bypass grafting play important roles in the process of clinical treatment,can effectively improve coronary circulation,promote reascularization,save a lot of patients with CHD,but the effect is still poor in patients with part of multivessel coronary artery lesions or refractory myocardial ischemia caused by repeated episodes even in patients with ischemic cardiomyopathy.Despite the ischemic myocardial was reperfused,but the ischemia-reperfusion injury can not be ignored.With the rapid development of genetic engineering technology,some scholars provide many new treatment strategies to use gene therapy for refractory myocardial ischemia.Nowadays,the transfection efficacy of non-virus gene vector need further improvement.The new methods have been explored constantly in biomedical field,for improving the targeted gene transfection efficiency in order to achieve comparable to viral vector transfection level,such as biotin-avidin system,antigen-antibody targeted combination,new type of nanometer microbubble,magnetic microbubble and so on.At present,these methods are limited to break through the barrier of cell membrane.In addition to the cell membrane,there is also nuclear envelope barrier in eukaryotic cells.Nuclear membrane is an important barrier for the transfection of exogenous gene.All materials must go through nuclear pore complexes into the nucleus.The small molecules with molecular weight<60 KD or diameter under 9 nm can be diffused through nuclear pore complexes passively,but the macromolecule material such as plasmid DNA must be approved by nuclear localization signal(NLS)peptide mediating the transport to enter the nucleus actively.The NLS with positively charged can be linked firstly with the negatively charged DNA by electrostatic adsorption,and then combined with microbubble formation a new nucleophilic carrier,which could play an important role in the transfection.Moreover,the effect of ultrasound targeted irradiation microbubble destruction(UTMD)as a non-virus carrier gene transfection method has been confirmed.Its main principle is that,the cavitation effect when the microbubble was destructed could increase the cell membrane permeability,and then it can make the exogenous gene enter into cells and transfect.On the basis of the UTMD could breakthrough the cell membrane,the NLS peptide was used as the nuclear wizard adding exogenous DNA into the nucleus.This study was performed on both of them to improve the transfection efficiency.NLS is mainly classified as the classic and non-classic two types.The classic nuclear localization signal(cNLS)peptide could play the nuclear transport functions not only associated the connection mode with DNA,but also closely related to the order of peptide sequence structure and the type of amino acid.The NLS with correct sequence structure can be combined with the nuclear import receptor.The cNLS link at the C-side of the good protein.When the NLS with error linked position or reverse sequence cannot be recognized by nuclear import receptor,lost the nuclear input function.In order to confirm the role of cNLS,some researchs also make the mutation of one basic amino acid in the cNLS sequence of 126PKKKRKV132,for example the 128th of lysine(K)could mutate into the threonine(T).The mutative nuclear localization signal(mNLS)peptide will not be able to play the nuclear import role.For further validation the role of cNLS in nuclear transport,some scholars adopt special material such as exogenous agglutination to close the nuclear pore,prevent the good protein with cNLS combined with the nuclear import receptor.Then the targeted gene will not be able to enter into the nuclear and play the corresponding role.The technology of ultrasound irradiation targeted microbubble destruction mediated-gene transfection mainly adopts the intravenous injection method.The research about intravenous ultrasound targeted microbubble destruction(IV-UTMD)has made certain progress,but the microbubble and targeted gene can get into left ventricle after venous system and pulmonary circulation,part of them pass through the aorta and distribute the whole body.At last the quantity of microbubble and gene reached the target tissue was less.That it is difficult to play a biological effect to a great extent.And after intracoronary artery injection,the targeted gene can make through collateral circulation capillary network to gather around in the targeted tissue,improving the content and concentration of the gene.Therefore,in this study we envisaged the first intracoronary injection microbubble,NLS and angiogenin 1(Angl)plasmid gathering the myocardial infarction(MI)surrounding area,then we made more gene enter into myocardial tissue for transfection through the cavitation effect of ultrasonic irradiation targeted microbubble destruction(IC-UTMD),promote angiogenesis,improve left ventricular remodeling.Meanwhile,the study about intramyocardial injection also made some progress.But in the previous the intramyocardial injection must open the chest firstly,that could induce the serious injury and infection.The intramyocardial injection under the guide of ultrasound can overcome the shortcoming,and proved to be safe and feasible.Therefore,on the basis of coronary artery injection,the comparison of the intramyocardial injection,intracoronary injection and intravenous injection in this study were performed for the efficacy and safety of kinds of treatments.Based on the above research background,the new nuclephilic carrier system was designed to help Angl gene transfection in this study.(1)the ultrasound microbubble was used as the gene carrier,adsorb with NLS through electrostatic interaction,form the new nucleophilic gene carrier,improve the efficiency of the exogenous gene into the cell cytoplasm and nucleus,while protecting the genes from the nuclease degradation,to improve the efficiency of gene transfection;(2)in order to further validate cNLS in the role of transfection,the mNLS and nuclear blocker of cNLS mediated gene into the nuclear were studied from the opposite side;(3)on the basis of NLS enhance gene into the nuclear for transfection in vitro,the intracoronary ultrasound targeted microbubble destruction(IC-UTMD)was used to increase the Angl gene content in MI surrounding area,make more can gene transfection in targeted area,promote angiogenesis and improve left ventricular remodeling;(4)The safety and efficacy were evaluated during the ultrasound guided intramyocardial injection,intracoronary injection and intravenous injection and them combination with UTMD.This study is divided into four parts.Part1The enhanced effect of nuclear localization signal peptide during the ultrasound targeted microbubbles destruction mediated gene transfectionObjective:To investigate the transfection efficiency combining ultrasound targeted microbubbles destruction(UTMD)and nuclear localization signal(NLS)peptide for facilitating the plasmid of enhanced green fluorescent protein(phAngl-EGFP)into nucleus.Methods:The vector of GV230 plasmid was used to construct the hAngl-EGFP plasmid and identificate it,a large quantity of plasmid was extracted after the success of the gene sequencing.The synthesis of NLS was detected by mass spectrometry and HPLC.The NLS was labeled by FITC,and the nucleic acid dye Cy3 was used to mark the hAngl-EGFP,the different mole ratio was adjusted between the NLS and hAngl-EGFP plasmid to observe the best ratio.The potential of the NLS-Angl combined with microbubble forming a nuclephilic gene carrier was detected by Zeta potentiometer.This study was divided into 3 groups,group A:UTMD+phAngl-EGFP;group B:UTMD+NLS+phAngl-EGFP;group C:Lipo3000+ phAngl-EGFP.The NLS was labeled by FITC and phAngl-EGFP was marked by Cy3.The different mole ratio was adjusted between NLS and phAngl-EGFP for observing the best ratio of combination.The human umbilical vein endothelial cells(HUVEC)were transfected by the optimum ultrasonic irradiation parameters and the optimal NLS/phAngl-EGFP mole ratio.6 h after transfection,the rate of Cy3 labeled pDNA into cells and nuclear were detected by flow cytometer and laser confocal microscope respectively.48 h after transfection,the transfection efficiency was detected by flow cytometer;the survival rate of cells was measured by CCK8.RT-PCR and Western blotting technology were used to detect the relative expression amount of mRNA and protein.The above indicators were compared in 3 groups,which were used to evaluate the enhanced effect of NLS in UTMD mediated gene transfection.Results:? Both the experiment of agarose gel electrophoresis and gene sequencing confirmed the hAngl-EGFP plasmid was successfully built.The purity of NLS was more than 98%.? 6 h after transfection,the NLS with green fluorescence and phAng1-EGFP with red fluorescence can show at the same site and signal intensity within the cell,that suggested a combination between them,agarose gel electrophoresis showed that the best molar ratio of NLS/phAngl-EGFP combining was 104:1.The potential of NLS-Angl nuclephilic gene carrier was +11.7?57.5 mV.? 6 h after transfection,the rate of phAngl-EGFP into the cells were(63±12)%,(80±10)%and(92±8)%.The rate of phAngl-EGFP into the nucleus were(17±3)%,(50±12)%and(35±8)%in 3 groups respectively(P<0.05).They were 1.3 and 2.9 times in group B than group A,1.5 and 2.1 times in group C than group A.? 48 h after transfection,the cell activity were>80%in all groups;the transfection efficiency,relative quantity of mRNA and protein expression were increased gradually.There were significant differences in 3 groups(P<0.05).They were 1.6,2.3 and 2.4 times in group B than group A,still lower than group C.Conclusions:The UTMD combining NLS can promote the phAngl-EGFP into nucleus for improving the transfection efficiency.The NLS peptide can play an enhanced effect as a new strategy of UTMD.Part 2 The study of ultrasound targeted microbubble destruction combinated classic nuclear localization signal peptide for facilitating gene transfectionObjective:To investigate the transfection efficiency of combining ultrasound targeted microbubble destruction(UTMD)and classic nuclear localization signal(cNLS)peptide for facilitating targeted gene into nucleus.Methods:The plasmid DNA(pDNA)was transfected into the 293T cells by different ways.This study was divided into four groups,control group:UTMD+pDNA;cNLS group:UTMD+pDNA+cNLS;mutative group:UTMD+pDNA+mNLS;and retardant WGA group:UTMD+pDNA+cNLS+WGA.The NLS was labeled by FITC and pEGFP was maked by Cy3.Six hours after transfection,the rate of plasmid into cells was detected as the percentage of Cy3 positive cells by flow cytometry,the rate of plasmid into nuclear was calculated as the percentage of Cy3 fluorescence intensity in nucleus to the whole cell by laser confocal microscope.48 hs after transfection,the transfection efficiency was detected by flow cytometry,the survival rate of cells was measured by CCK8.RT-PCR and Western blotting were used to detect the relative expression amount of mRNA and protein.The differences were compared in four groups to evaluate the enhancement effects of cNLS during UTMD mediated gene transfection.Results:?Six hours after transfection,almost all green fluorescence showed in the nucleus in cNLS group,it appeared in both the cytoplasm and nucleus in mNLS group,but mostly appeared in the cytoplasm and almost none in nucleus in WGA group.? Six hours after transfection,the rate of pEGFP into the cells were(61±11)%,(80±10)%,(55±9)%and(58±10)%;the rate of pEGFP into the nucleus were(20±4)%,(50±11)%,(18±3)%and(10±3)%in four groups respectively.The rates were highest in cNLS group and lowest in WGA group,the difference was statistically significant(P<0.05).? Forty-eight hours after transfection,the cell activity were>80%in all groups;the transfection efficiency,the relative quantity of mRNA and protein were highest in cNLS group and lowest in WGA group,the difference was statistically significant(P<0.05).Conclusions:The UTMD combining cNLS can promote pEGFP into the nucleus for improving the transfection efficiency.The mNLS and WGA could reduce the transfection efficiency.Part 3 The intracoronary ultrasound targeted microbubble destruction technique enhanced the angiogenesis effect in canine after acute myocardial infarctionObjective:To evaluate the angiogenesis effect by intracoronary ultrasound targeted microbubble destruction(IC-UTMD)technique mediated the NLS-Angl nucleophilic gene carrier in canine after acute myocardial infarction(MI).Methods:21 dogs were established successfully MI model by trans-catheter coronary artery embolism,and divided into:control group(n=7,intracoronary injected saline),the non-nuclephilic group(n=7,the Ang1 plasmid and microbubble were injected intracoronary),nuclephilic group(n=7,the NLS-Angl plasmid and microbubble were injected intracoronary).The echocardiography and myocardial contrast echocardiography were performed before infarction,1 hour after infarction,and 1 month after treatment.One month later,TTC and Evans blue staining were performed to evaluate the percentage of infarction area.Masson staining was used to assess the fibrosis of the infarction myocardial,the immunohistochemical of CD31 could calculate the density of capillary.The tissue ice slice was used to observe the expression of EGFP green fluorescent protein,RT-PCR and Western blotting detected the relative expression of the hAngl-EGFP mRNA and protein.The infarction percentage,angiogenesis effect and transfection efficiency were compared between groups.And the relative expression of calcium ion operation key protein sarcoplasmic reticulum Ca2+-ATPase 2a(SERCA2a)and phospholamban(PLB)were detected by Western blotting.The myocardial ultrastructure changes were observed by transmission electron microscopy.Results:?The comparison of conventional echocardiographic parameters,both the LVEDD and LVESD showed a increased trend before myocardial infarction,1 hour after MI,and 1 month after treatment.The LVESD was smaller in the nucleophilic carrier group than in the other groups,the LVEF was higher in the nuclephilic group than in the other groups,the difference was statistically significant(P<0.05);? the myocardial contrast echocardiography showed that the intensity ratio of infracted region/normal region and the increased rate of intensity were both higher in the nuclephilic group than in the other groups(P<0.05).?The TTC and Evans blue staining showed reduced relative infarction range and Masson's staining showed decreased collagen fiber in the nuclephilic group;A higher blood vessel density was detected by CD31 immunohistochemical staining in the nuclephilic group than in the other groups(all P<0.05).?The comparison of transfection efficiency,the marker protein of EGFP showed more green fluorescent expression by frozen section in the nuclephilic group;The relative quantity of mRNA and protein of Angl were both higher in the nuclephilic group than in the other groups by RT-PCR and Western blotting,the difference was statistically significant(all P<0.05).? The relative expression of SERCA2a was increased from the control group,the non-nucleophilic group to the nucleophilic group,PLB was just the opposite with SERCA2a.There was significant difference between them(P<0.05).Conclusions:The nuclephilic gene carrier of NLS-Angl combined the IC-UTMD can improve the transfection efficacy of hAngl in myocardial,promote angiogenesis,reduce the area of MI,and improve the left ventricle function after MI.Part 4 The effect of ultrasound guided via chest puncture intramyocardial injection and ultrasound irradiation targeted microbubble destruction(UTMD)-mediated the Angl gene therapy in myocardial infarction caninesObjective:To evaluate the safety and effect of ultrasound guided via chest puncture myocardial injection and UTMD-mediated the angiogenin 1(Angl)gene therapy in myocardial infarction(MI)canines.Methods:52 dogs were divided into four groups(n=13 in each group):the control group(MI dogs without treatment);the intramyocardial injection group(MI dogs with intramyocardial injection and UTMD treatment);the intracoronary injection group(MI dogs with intracoronary injection and UTMD treatment);and the intravenous injection group(MI dogs with intravenous injection and UTMD treatment).4 weeks later,the safety and incidence of complications were compared.The dimensions and systolic function of left ventricular were measured by echocardiography.The distribution of FITC labeled plasmid in myocardial tissue was detected by frozen section.The collagen fiber percentage was assessed by Masson's staining.The immunohistochemical of CD31 and a-SMA was applied for quantifying the capillary and arteriolar density,respectively.The relative quantity of mRNA and protein of Angl were detected by RT-PCR and Western blotting respectively.The concentration of serum troponin(cTnI)and N terminal B-type natriuretic peptide(NT-proBNP)were determined at different time point.Results:?4 weeks later,the survival and complications showed no significant differences during four groups(P>0.05).?The distribution of the FITC green fluorescence in the myocardial tissue was most in the intramyocardial injection group,followed by the intracoronary artery injection group(P<0.05);?The comparison of conventional ultrasonic parameters,the lower LVESD and higher LVEF and FS was in the intramyocardial injection group than the control group,but lower than intracoronary artery injection group,the difference was statistically significant(P<0.05);?The Masson's staining showed lower collagen fiber in the intramyocardial injection group than the control group.And there was no statistically significant difference between the intramyocardial injection group and the intracoronary artery injection group(P>0.05).A higher blood vessel density was detected by CD31 and a-SMA immunohistochemical staining in the intramyocardial injection group and the intracoronary artery injection group(P<0.05)? The relative quantity of mRNA and protein of Angl were both higher in the intracoronary artery injection group by RT-PCR and Western blotting respectively,followed by intramyocardial injection group,both was higher than in the control group,the difference was statistically significant(all P<0.05).? The peak of cTnI was at 24 hours after myocardial infarction,the treatment groups were higher than the control group.The NT-proBNP in each treatment group began to reduce gradually from 7 days after MI,to 14 days and 1 month later the NT-proBNP concentration was significantly lower in the intramyocardial injection group and the intracoronary artery injection group than in the control group and intravenous injection group,the difference was statistically significant(P<0.05).The concentration of serum cTnI and the NT-proBNP at each time point showed no significant differences between the intramyocardial injection group and the intracoronary artery injection group(P>0.05).Conclusions:the ultrasound guided via chest puncture intramyocardial injection is a safe and effective way of gene transfection and its mediated Angl transfection can promote angiogenesis surrounding the infarcted myocardium,reduce the degree of fibrosis.But the higher transfection efficiency,reversing left ventricular remodeling,and improving left ventricular function are more significant in the intracoronary artery injection group. |
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