Objective To study the anti-depressive mechanisms of acupuncture by investigating the effects of acupuncture on depressive behaviors and amino-neurotransmitter in hippocampus in rats treated with chronic mild unpredicted stresses.Methods Thirty adult SD rats were randomly divided into a normal group,a depressive model group and an acupuncture group,with 10 rats in each group.Separated feeding,long-term unpredictable and middle stimulation stress were used for development of depression rat model.At the same time,the third group was treated by acupuncture.Autonomic behavior was measured with Open Field test and concentration of amino-neurotransmitter in hippocampus was measured by high performance liquid chromatography coupled with fluorescence detection.Results Compared with the normal group,autonomic activity of the rats and the concentration of GABA were significantly reduced in the model group,while the concentration of Glu was significantly increased(P0.05).Conclusion Acupuncture could reverse the depressive behaviors of rats caused by middle stimulation stress,and its anti-depressive mechanisms is related to the function of regulating the concentration of amino-neurotransmitter in hippocampus.

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 determine whether it could enhance gene delivery and tumor transfection in vivo by combination of ultrasound-targeted microbubble destruction(UTMD)with polyethylenimine(PEI)in tumor xenografts.Methods Two different reporter plasmid[lueiferase(pCMV-LUC)and red fluorescent protein(RFP)]were incubated with PEI to prepare cationic compound(PEI/DNA)in various nitrogen:phosphate ratios(N/P ratios,nmol of nitrogen in the PEI/nmol of phosphate in DNA).Formation of PEI/DNA complexes were confirmed by the gel retardation assay.Human cervical carcinoma(Hela)tumors were planted subcutaneously in both flanks of female nude mice.Tumor-bearing mice were administered by tail vein with PBS,plasmid,plasmid and Sono Vue microbubble,PEI/DNA and Sono Vue microbubble.One tumor was exposed to ultrasound irradiation (3 MHz,2 W/cm2,2 min exposure,duty cycle 20%),while the other served as control.The feasibility of targeted delivery and tissue specificity facilitated by UTMD and PEI was investigated.The mice were sacrificed 3 days after ultrasound exposure.Tissue specimens were viewed with microscopy to determine the presence of RFP expression.The efficiencies of luciferase transgene expression were determined.Histology analysis was detected.Results Electrophoresis experiment revealed that PEI was mixed with plasmid to condense DNA efficiently.The application of UTMD significantly increases the tissue transfection in vivo compared to plasmid alone.RFP expression was present in all sections of tumors that received ultrasound exposure but not in control tumors.Results of luciferase activity showed that the expression of luciferase was to be 14 times greater in ultrasound-exposed tumors(P＜0.01).More importantly,the increase in transgene expresgion was related to UTMD with the presence of PEI dramatically.At least 10-fold increase of luciferase gene transfer was obtained in irradiated tumors compared to non-irradiated controls(P＜0.01),111-fold increase compared to UTMD alone(P＜0.01).There was not significantly gene expression in other organs or tissues regardless of US exposure(P＞0.05).No tissue damage was seen histologically.Conclusions The combination of UTMD with PEI can enhance targeted delivery and expression of reporter gene to tumors at intravenous administration.It is a promising new and safe method for gene delivery in vivo.

Objective To investigate different ultrasound parameters and transfection conditions that would affect transfection rate of red fluorescent protein(RFP)and cell viability of cancer cells.Methods In this study,Hela cells were cultured using two different protocols:(A)24 h culture for complete adherence;(S)suspension.Subsequently,cells were transfected following different ultrasound exposure protocols[1.0W/cm2;duty cycle(DC):10%,20%and 50%;exposure 1min or 3 min].Gene transfection and cell viability were evaluated.Treatment parameters optimized in Hela cells were applied for delivery RFP in 4 other cell lines(HepG2,Ishikawa,MCF-7 and B16-F10).Results Cell injury were found to increase progressively with DC and exposure time in group A.Cell detachment was significantly accompanied by ultrasound exposure in adherent HeLa cells.Cells in group S were found more prone to be transfected than group A with the same ultrasound parameters,while the survival rate was not decreased apparently.The ideal ultrasound conditions were noted to be at 1.0 W/cm2 irradiated 3 min with 20%DC using suspended protocol,producing maximum efficiency[transfection=(28.04±2.27)%]in gene delivery with minimum cell toxicity[cell viability=(81.20±1.73)%].These experiments also revealed different response to ultrasound treatment,but for all tested cell lines,dead and transfected cells in the treated groups were significantly different from the non-irradiated groups.Conclusions Ultrasound parameters and transfection conditions have a great impact on the gene delivery and cell viability.Gene delivery of ultrasound-mediated microbubble enhance should be optimized to improve the efficiency.

Objective To study the optimized condition of transfection efficiency for MCF-7 cells enhanced by ultrasound(US) irradiation and contrast agent combined with polyethyleneimine(PEI) and observe whether the combination can have a synergistic effect to increase DNA transfection. Methods MCF-7 cells were transfected with the compounds prepared by the vector of plasmid DNA encoding luciferase (pCMV-luciferase-GL3) and PEI.SonoVue microbubble was added to the cell suspension to serve as nucleation sites for aeoustic cavitation before US irradiation. The DNA expression of luciferase plasmid and viability of cells were evaluated. The strategy of US irradiation was optimized. Furthermore, the influencing factor, such as the concentration of plasmid, incubation time, serum, the type of solvent and the volume of culture media, were examined. Results The viability of cells and US-induced enhancement of luciferase activity were influenced by the US intensity,exposure time and duty cycle.US irradiation under an appropriate condition enables ceils to accelerate the permeation of the PEI/DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. Optimal US condition for the enhancement was determined to be 1 W/cm2,10% DC for 3 min. In contrast to the PEI/DNA complex alone without US irradiation or US irradiation alone, the combination of US irradiation with contrast agent and PEI had a significantly enhanced luciferase activity (P<0.01). The 2 h pre-irradiation incubation with PEI/DNA complex for MCF-7 ceils exhibited a significantly enhanced lueiferase activity (P<0.01). Besides,serum,type of solvent and the volume of culture media did affect the transfection efficiency. Conclusions The optimized parameters of US and transfection provide efficient gene delivery in MCF-7 cancer cells. The combination of US irradiation with contrast agent and PEI has a synergistic effect to increase DNA transfection. This is a simple and promising method to enhance the gene expression of plasmid DNA.

AIM: To validate the alternations of flow velocity patterns in the infarct-related artery (IRA) during no-reflow phenomenon in a canine model of acute myocardial ischemia and reperfusion by transthoracic Doppler echocardiography (TTDE) combined with myocardial contrast echocardiography (MCE) by means of administration of Albunex. METHODS: Nineteen dogs first underwent 60 min myocardial ischemia and then followed by 60 min,120 min and 180 min reperfusion ( n = 6, 6 and 7, respectively). The perfusion defect area determined by MCE at 60 min myocardial ischemia was regarded as risk area (RAMCE). The perfusion defect area defined by MCE after reperfusion was considered as no-reflow area (NRAMCE). The ratio between NRAMCE and RAMCE ≥ 25 %was regarded as the development of no-reflow phenomenon and the ratio of NRAMCE to RAMCE＜25% was considered as the myocardial reflow. The coronary flow velocity parameters in IRA were determined through TTDE. RESULTS: Two dogs died during experiment and the remaining seventeen dogs completed throughout the procedure.There were seven dogs in reflow group and ten dogs in noreflow group. No significant difference was present in reflow group between at baseline and at 60 min reperfusion in systolic peak velocity (PVs), systolic velocity time integral (VT Is), corrected systolic flow duration (cFDs),diastolic peak velocity (PVd), diastolic velocity time integral (VT Id), corrected diastolic flow duration (cFDd),diastolic deceleration rate (DDR), corrected diastolic deceleration duration (cDDD) (P＞0.05), however, a significant difference was found in no-reflow group between at baseline and at 60 min reperfusion in PVs,VTIs, cFDs, PVd, VTId and cFDd (P＜0.05). The most marked alterations during diastolic phase were the increase of DDR and reduction of cDDD. CONCLUSION: The impaired microvasculature may profoundly affect the coronary flow velocity pattern in the IRA. The increase in microvascular resistance and decrease in coronary perfused pressure can contribute to the changes.Transthoracic Doppler echocardiography combined with MCE has the capability of noninvasive assessment of coronary flow velocity pattern in the IRA during no-reflow phenomenon.

Objective Investigate the relationship between thromboelastography and coagulation tests,and evaluate of two kinds of methods in guiding significance in cardiac surgery patients perioperative blood transfusion.Methods Analysis of 108 patients with cardiac surgery in September 2014-August 2016 treated in our hospitalwere randomLy divided into TEG detection guide blood transfusion group (experimental group) and coagulation test guide blood transfusion group (control group),Respectively on two groups of blood transfusion rate,different components of blood transfusion and transfusion effect comparison.Results Through the comparison of the heart surgery perioperative blood use rate guided by TEG and coagulation test,found the transfusion rate of experimental group guide cryoprecipitate 25.9％ and fresh frozen plasma(FFP) 33.3％ was significantly lower than the control group 68.5％,however,platelet transfusion rate was significantly higher than the control group of 57.4％ to 20.4％.There was no statistically significant difference of red cell suspension (RCS) infusion rate between two groups,but the RCS infusion quantity of experimental group(3.6+ 1.6) was clearly lower than the control group (5.8+ 1.9),and the FFP and cryoprecipitate infusion quantity of experimental group was obviously lower than control group(P＜0.05),too.Experimental groups of intraoperative blood loss,postoperative blood loss and rebleeding rate was lower than control group (P＜ 0.05).Concltsion That TEG guide cardiac surgery patients perioperative blood transfusion can reduce the use amount of blood products,and the prognosis of disease outcome is better than that of routine coagulation tests guidance.TEG can reduce the surgical complications and the bleeding amount,and it has great sense to guide clinical composition blood transfusion,so it is worth promoting.

Objective To investigate different pulsed ultrasound (PUS) parameters and culture conditionsthat would affect cell viability and sonoporation on cell membrane of human cervical cancer cells (HeLa). MethodsHeLa cells were cultured in two different conditions ( in suspension or in monolayer). Cells were exposed to differentPUS intensity (0.4 W/cm2, 1.0 W/cm2, 1.6 W/cm2, 2.2 W/cm2), duty cycle (10%, 20%, 50%) and expo-sure time ( 1 min or 3 min). Cell viability was analyzed by flow cytometry. Using microscope and scanning electronmicroscopy (SEM) , the changes of shape and the sonoporation on cell membrane induced by PUS were observed.Results Low intensity and duty cycle did not exert a great impact on the cell viability. Cell injury was found to in-crease progressively with high intensity ( 1.6 W/cm2 , 2.2 W/cm2 ) and duty cycle ( 50% ) ( P < 0. 01 ) , and celldetachment was significantly accompanied by PUS exposure in adherent HeLa cells. Results of factorial design showedthat the culture conditions and the PUS parameters had significant interaction ( P < 0.01 ). SEM demonstrated insome detail the phenomenon of transient pores in the cell membrane under suitable PUS irradiation. The ideal sonopo-ration conditions that cell viability was above 80% and more membrane holes were noted to be at 1.0 W/cm2 expo-sure for 3 min with a duty cycle of 20% in cell suspension. Conclusion The optimized conditions of the PUS pa-rameters and the culture conditions could lower the cell injury and exert a great impact on the sonoporation. It couldproduce remarkable membrane pores on cells and enhance cell membrane permeability, which facilitate transportationof macromolecules into cells.

Objective To explore left ventricular remodeling index(LVRI),systolic function and their correlation by real-time three-dimensional echocardiography(RT-3DE)in patients with coronary artery disease(CAD).Methods According to coronary artery angiography,one main vessel stenosis no less than 50% was defined as positive standard,RT-3DE data of 24 patients with left circumflex coronary artery or/and right coronary artery stenosis(LCA/RCA group),21 patients with left anterior descending coronary artery stenosis(LAD group),27 patients with double or triplex coronary arteries stenosis which must included LAD(vessels group)and 22 normal controls(NC group)were acquired.Left ventricular end-diastolic volume(EDV),left ventricular end-diastolic epicardial volume(EDVepi),left ventricular ejection fraction(EF)were automatically measured by RT-3DE analysis software;left ventricular mass[1.05×(EDVepi-EDV)],LVRI(left ventricular mass/EDV)were calculated.The inter-group differences of LVRI,EF and intra-group correlations between LVRI and EF were analyzed.Results LVRI and EF significantly decreased in order(P＜0.05 or P＜0.01);and their correlation coefficents were r=0.10(P＞0.05),0.86(P＜0.01),0.83(P＜0.01)and 0.77(P＜0.01)in NC group,LCA/RCA group,LAD group,vessels group,respectively.Conclusions LVRI measured by RT-3DE can assess left ventricular remodeling,and reflect left ventricular systolic function in different CAD,which can be as a new approach for evaluating left ventricular remodeling in clinic.

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.