Objective To study the transfeetion efficiency and safety of liposome microbubble(LM)on red fluorescent protein(RFP)in vitro and in vivo under ultrasound mediated gene transfection(USMGT)conditions.Methods Plasmids containing RFP were added to cultured Hela cells followed by ultrasound (US)exposure with LM.Different concentration of LM,US intensity and exposure time were optimized.Transfection efficiency was evaluated by fluorescent microscopy and FACS.Cell viability was verified by propidium iodide assay.In transplanted tumors in vivo study,LM and plasmid(P)were injected into the nude mice followed by US exposure(P+LM+US group).Nude mice undergoing plasmid injection alone(P group),plasmid injection and US exposure(P+US group)and plasmid and LM injection(P+LM group)were used as controls.Frozen section and histological examination were conducted and RFP expression was evaluated.Results LM and US exposure significantly increased transfeetion efficiency in cultured Hela cells (P＜ 0.01).Transfection efficiency was the most prominent under the condition of US intensity of 1.0 W/cm2 with 6%LM,duration 3 min.No apparent cell damage was found in the all groups.In transplanted tumors,strong RFP was seen in P+LM+US group.It was significantly higher than in any other groups(P＜0.0 1).No tissue damage was seen histologically.Conclusions LM could enhance USMGT effectively without causing any apparently adverse effect in vitro and in vivo.This method would be a novel,effective,safe non-viral gene transfection method and provide an alternative to current clinical gene therapy.

Objective To investigate the value of time parameters(the transit time in coronary and the half filling time in myocardium)in assessing the blood flow of ischemia myocardium through establishing canine model of acute myocardial infarction.Methods Eighteen healthy anesthetized open-chest dogs were ligated the left anterior descending(LAD),3 hours later the contrast agent(C3F8)was injected into femoral vein in a fixed velocity to perform myocardial contrast echocardiography(MCE)examination.The first perfusion and the replenishment processes were recorded by real-time tri-plane MCE(RT-TP-MCE)and real-time single-plane MCE(RT-SP-MCE)respectively.both the two different processes were analyzed by the function Y=A ×(1-e-βt)+B.RT-SP-MCE used A(plat of peak intensity)and β(slope rate of the curve)values to quantitatively calculate the myocardial blood flow(MBF=A×β).The time point of the first microbubbles coming into the left ventricle was normalized to O in RT-TP-MCE,the time point of the first microbubbles coming into the myocardium was defined as the transit time in coronary(Tc),and the time point when microbubbles in myocardium achieved the 50% plat video intensity was defined as the half filling time in myocardium(Tm).Evans blue dye and TTC staining were performed to identify normal.ischemic and infarct myocardium.Results The time parameters of the two groups-normal myocardium and ischemic myocardium from RT-TP-MCE were:Tc(10.1±1.3)s and(20.4±7.1)s(P＜0.01),Tm(17.1±2.2)s and(39.7±8.8)s(P＜0.01).There were significantly negative correlation between the time parameters(Tc,Tm)and MBF from RT-SP-MCE in ischemic myocardium(r=-0.876,P＜0.01;r2=-0.894,P＜0.01).Conclusions The first perfusion imaging with RT-TP-MCE could be used to simultaneously,quantitatively evaluate the myocardial perfusion.The lower flow,the longer transit time in coronary and half filling time in ischemic myocardium.

Objective To evaluate the collateral circulation after canine acute coronary artery occlusion with contrast real-time three-dimensional echocardiography (RT-3DE). Methods Left anterior descending coronary artery was ligated in 12 healthy mongrel dogs. All dogs underwent myocardial contrast echocardiography (MCE) before ligation, immediately after ligation, at 30 min and 180 min after ligation respectively, and myocardial mass with collateral supply was calculated. Results Different degrees of collateral circulation were established in all dogs after left anterior descending coronary artery ligation. At 30 min after ligation, the myocardial mass with collateral supply was (9.65±2.90) g, while at 180 min after ligation were (12.58±3.98) g (P<0.01). Conclusion The coronary collateral circulation can be observed clearly and myocardial mass with collateral supply can be quantified accurately with contrast RT-3DE.

Objective To assess longitudinal, radial and circumferential systolic strain of regional myocardium in patients with coronary artery disease by 2-dimensional strain echocardiography, and to explore stenosis of coronary artery impacting on regional left ventricular systolic dysfunction in quietscent condition. Methods Twenty-six patients with coronary artery disease were entrolled into this study and 26 healthy volenteers in the control group. The two-dimensional loop-einec were obtained in apical 4-chamber view,apical 2-chamber view and long axis view of left venricular, and three levels of the short axis views (mitral valve,papillary muscle and cardiac apex). According to left ventricle 18 segments divla, there were 194 affected myocardiums in 26 cases. According to stenosis degrees of coronary artery, the affected myocardiums were divided into two groups: group A (coronary stenosis degree≤70%) and group B (coronary stenosis degree>70%). Longitudinal, radial and circumferential systolic strain (SL, SR, SC) from regional myocardium were analyzed. Results Group A had 90 segments. Compared with control group,SL was decreased significantly from control group (P <0.05),while there were no significantly differences in SC and SR ( P>0.05). Group B had 104 segments. Compared with control group, SL, SC and SR were decreased significantly in group B(P <0.05). There were significant differences in SL,SC and SR between group A and group B(P<0.05). Conclusions Systolic dysfunction of left ventrieular regional myocardium in patients with coronary artery disease could be accuratly analyzed by 2-dimensional strain echocardiography. Changes of SL,SC and SR of regional myoeardium could reflect coronary stenosis severity degree.

Objective To investigate the characteristics and the enhanced patterns of carotid plaque using contrast-enhanced ultrasound (CEUS) and patients with cerebral infarction.Methods The patients with carotid plaque were divided into two groups according to whether they had cerebral infarction:54 patients (62 plaques with CEUS) with cerebral infarction were included in group A,and 48 patients (54 plaques with CEUS) without cerebral infarction were included in group B.The plaques were divided into four grades according to the degree of plaque enhancement.According to the source of intraplaque contrast agents,plaque enhancement patterns were divided into adventitia enhancement,lumen enhancement and mixed enhancement.To analyze the degree and pattern of carotid plaque enhancement in the two groups.Results Carotid plaque enhancement in cerebral infarction group was mainly grade 3 (26/62) and grade 4 (22/62),while that in non-cerebral infarction group was mainly grade 2 (20/54) and grade 3 (20/54).There was significant difference between the two groups in the proportion of carotid plaque enhancement of grade 2 (P =0.019) and grade 4 (P =0.041).The proportion of plaque adventitia enhancement model in group A(27/59) was lower than that in group B (37/50),with statistically significant difference (P =0.003).While the proportion of mixed enhancement mode in group A was significantly higher than that in group B (P =0.003).Conclusions The enhancement of carotid plaque was obvious in cerebral infarction patients,and the mixed enhancement pattern was more common.It suggested that the communication between vascular cavity and plaque might be an important factor leading to cerebral infarction.

Objective To study on the relationship between the serum level of highsensitivity c-reaction protein (hs-CR),matrix metalloproteinase 9 (MMT-9),tissue inhibitor of metalloproteinase 1 (TIMP-1) and carotid plaque stability in elderly people.Methods According to the carotid ultrasonography examination,120 old people were divided into vulnerable plaque group (group A,n =45),stable plaque group (group B,n =41) and no plaque group (group C,n =34),and serum levels of hs-CRP,MMP-9,TIMP-1 in each group were also detected.Results The serum levels of hs-CRP and MMP-9 of unstable plaque group and stable plaque group were higher than those of non-plaque group (P ＜ 0.05).hsCRP and MMP-9 were positively correlated with carotid plaque unstability,while TIMP-1 was negatively correlated with carotid plaque unstability,hs-CRP,MMP-9 and TIMP-1 were independent risk factors for carotid plaque stability.Conclusions Serum levels of hs-CRP,MMP-9 and TIMP-1 are closely related to the stability of carotid plaque.Elevated levels of hs-CRP and MMP-9 increase the risk of carotid plaque,and elevated levels of TIMP-1 decrease the risk of carotid plaque.

Summary: The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein (EGFP) gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction (UTMD) and polyethylenimine (PEI). SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.