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.

Objective To evaluate the correlation between hyperlipidemia and lower limb arterial disease in eldedy diabetic patients with color Doppler uhrasonography.Methods Two hundred and sixty elderly diabetic patients and 206 normal subjects were enrolled in the study.Doppler uhrasonographic manifestations of lower limb arterial disease,blood pressure and blood biochemistry parameters including fasting plasma glucose(FPG),postprandial plasma glucose(PPG),TC,TG,HLD-C,LDL-C,apoprotein B (apoB),very low density lipoprotein cholesterol(VLDL-C),apoprotein Al(apoA1)and fibrinogen(Fb)were compared between the 2 groups.Results (1)The blood biochemistry parameters,including the levels of FPG,PPG,TC,TG,VLDL-C,LDL-C,apoB and Fb were higher in diabetic patients t11an in normal subiects.while HDL-C was lower(all P＜0.05).(2)The thickness of intima-media(IMT)in the lower limb arterial disease group was significantly increased and the presence of arterial stenosis and wall plaques more in diabetic patients[IMT in 205 cases(78.85%),atherosclerotic plaque in 241 cases(92.69%),muhivessel disease(more than 2 vessels)in 60 cases(23.08%)and arterial stenosis in 89 cases (34.23%)]than in normal subjects[IMT in 37 cases(17.96%),atherosclerotic plaque in 51 cases (24.76%),muhivessel disease in 27 cases(13.11%)and arterial stenosis in 3 cases(0.01%)],all P＜0.01.(3)Regression analysis showed that lower limb arterial disease was correlated with high level of LDLC and low level of HDL-C.Conclusions Arteriosclerotic plaque is one of the early manifestations in elderly diabetic patients and hyperlipidemia may provide the basis of its formation.Doppler uhrasonographic examination of lower limbarterial discloses the early arteriosclerotic changes of lower limb arterial disease in elderly patients and offers the basic data of indication for clinical treatment.

Objective To explore effects of hcmoperfusion on toxic ingredients in plasma of rabbiis with a-cute intoxication of Radix Aconiti Kusmezoffii Monkshood.Method Sixteen male Japanese Giant Ear Rabbits were randomly divided into acute poisoning(AP)group and acute poisoning + hemoperfusion(AH)group(8 an-hnals in each group).Acute poisoning models were established in rabbits of both groups with intragastric adminis-tration of Radix Aconiti Kusmezoffii Monkshood liquid in dose of 1 mL/kg in order to produce arrhythm which oc-curred within ode hour after intragastric administration was regarded as the criteria of successful animal model.and then hemoperfusion with active carbon was performed for 2 hours in AH group.The pathological chanses of brain,myocardium and hepatic tissues were observed.The plasma concentrations of toxicants including mesaconitine,a-conitine and hypaconitine were measured by using HPLC-MS at 1 h,2 h,3 h,and 6 h after poisoning.Student's T test was used to identify the significance.Results The brain.myocardium and hepatic tissues of the rabbits in AP group showed hyperemia and edema which were attenuated after hemoperfusion.The plasma concentrations of mesaconitine,aconitine and hypaconitine revealed no significant differences between AP group and AH group with-in one hour after poisoning(P＞0.05),while at 2 h and 3 h after poisoning,the plasma concentrations of mesaconitine were(2.11±1.08)ng/mL,(2.02±1.46)ng/mL,respectively,aconitine(39.70±9.31)ng/mL,(19.71±16.06)ng/mL,respectively,and hypaconitine(1.70±0.71)ng/mL,(2.12±1.33)ng/mL,respec-fively in AH group,and they were significantly lower than those in AP group(P＜0.05).Conclusions The the plasma concentrations of mesaconitine,aconitine and hypaconitine were lower and the histopathological changes were attenuated after hemoperfusion.Hemoperfusion is a good intervention for acute intoxication of Radix Aconiti Kusmezoffii Monkshood.

Objective To evaluate the correlation of hyperlipidemia and carotid arthrosclerosis manifestations on color Doppler ultrasonography and dynamics of elderly diabetes patients. Methods One hundred and fifty elderly diabetes patients and 106 normal subjects were enrolled in the study. The carotid color Doppler ultrasonography and blood sugar, TC, TG, HDL-C, apoB,VLDL, apoA I, apoB were examined and compared between the two group. Results The blood sugar, TC, TG, HDL-C, apoB, VLDL, apoA I, apoB were increased in diabetes patients which were higher than in normal subjects (P

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 investigate the expression pattern of resistin in subcutaneous adipose tissue and its effect on glycometabolism in rats with traumatic brain injury (TBI).Methods Fifty-six SD rats were randomly divided into TBI group (n =48) and control group (n =8) according to the random number table.mRNA and protein expressions of resistin in subcutaneous adipose tissue were detected with real-time PCR and Western-blot.Concentrations of serum insulin and serum fasting blood glucose were evaluated using the ELISA method and quantitative estimation of insulin sensitivity was performed.Indices measured and their correlations were statistically analyzed.Results Levels of the resistin,serum insulin and FBG were significantly higher in TBI group than in control group (P ＜ 0.05).Quantitative estimation of insulin sensitivity lowered in TBI group compared to control group (P ＜ 0.05).Single factor linear correlation analysis showed negative correlation between resistin expression and quantitative insulin sensitivity check index in TBI group (P ＜ 0.05).Conclusions Resistin is shown to have significant expression in subcutaneous adipose tissue and involve in glycometabolism.Obviously,resistin may play a significant role in insulin resistance after TBI in rats.

BACKGROUND:Recently,the development of tissue-engineered technique lms broadened the study of artificial esophagus.Some investigators have inoculated esophageal epithelial cells cultured in vitro onto compound polymer material and successfully constructed tissue-engineered esophagus.OBJECTIVE:To investigate the feasibility of tissue-engineered artificial esophagus by combining dog esophageal epithelail cells and an acelhilarized porcine thoracic aorta allogenic matrix.DESIGN:An experimental observation.SETTING:Central Laboratory,Taishan Medical College.MATERIALS:This study was carried out in the Central Laboratory.Talshan Medical College from June to December in 2004.Three hybrid dogs,24-hour-old,were provided by the Laboratory Animal Center,Taishan Medical College.The protocol was performed in accordance with ethical guidelines for the use and care of animals.The experimenhal instruments and reagents were as follows:CO2 incubator(MCO-15AC,SANYO),hypothermal high-speed centrifuge(RC-26,Dupont),trypsin,transferrin,type Ⅱ collagenase(Gibco),dulbecco's modified eagle's medium(DMEM),DispaseⅡ isolated enzyme,and rat monoclonal anti-keratin antibody(Sigma).METHODS:Acellularization of porcine aortas was performed by a method of enzyme-detergent.Esophageal epithelial cells of hybrid dogs were in vitro isolated,cultured and proliferated.Next,they were inoculated onto an acellularized porcine thoracic aortas allogenic matrix seaffold.Three and seven days later,the growth of esophageal epithelial cells on the acellularized matrix was observed under an electron microscope.MAIN OUTCOME MEASURES:Morphology of esophageal epithelial cells cultured in vitro;Biocompatibility of acellular matrix and dog esophageal epithelial cells.RESULTS:The acellularized procedure resulted in an almost complere removal of the cells and the loose three-dimensional matrix.The acellular matrix could be reseeded with expended esophageal epithelial cells in vitro.and esophageal epithelial cells had the potential of spread and proliferation.CONCLUSION:Acellular matrix possesses satisfactory biocompatibility for allogenic esophageal epithelial cells.Tissue-engineered artificial esophagus can be generated in vitro by a combination of esophageal epithelial cells and allogenic aceilularized matrix.

This article was aimed to study the antioxidant and antihyperlipidemic effect of total phenolic composition prepared from pineapple (A nanas comosus L.) leave. It had a rapid absorption after intragastric administration and the principle ingredient, p-coumaric acid, reached the peak concentration after 5 minutes of administration. The mice model with single intragastric administration of lipid and glucose were used in the observation of changes on postprandial glucose, lipid and intestinal lipase at different time points after drug administration. The results showed that phenols of pineapple leave can inhibit triglyceride and glucose absorption in certain extent. No significant effect was observed on inhibition at 30 minutes after the phenol administration. However, the intestinal lipase activity was obviously inhibited in normal mice and the intestinal lipase activity decline caused by acute lipid consumption can be reversed. It was concluded that the phenols pineapple leave may inhibit the absorption of lipids with correlation to lipase activity. It had certain regulation effect on the high postprandial glucose and fat absorption.

Objective:To investigate the impact of human umbilical cord-derived mesenchymal stem cells on the activation ,the survival of human peripheral blood mononuclear cell ( hPBMC) and the proportions of each human lymphoid subgroup .Methods:PB-MC were isolated from healthy donors by density gradient centrifugation , then cultured in MSC-CM as treatment group after being acti-vated by OKT3.Each lymphoid subgroup proportion was analyzed by flow cytometry to observe the difference between treatment and control group .The effect of MSC-CM on activated PBMC for the production of IFN-γand IL-10 were tested by ELISA .The level of ap-optosis was assessed by flow cytometry with Annexin-V/PI as fluorescent marker .Results:Compared with the control group , MSC-CM down-regulated the ratio of CD4 +T cell to CD8 +T cell, and increased the proportion of CD4 +CD25 +CD127low Treg cell, thus other subgroup had no significant difference .MSC-CM inhibited the production of IFN-γby PBMC, but promoted the secretion of IL-10, and protected PBMCs from apoptosis when activated with OKT 3.Conclusion:hUC-MSC may play a role of immunosuppression by promo-ting the proliferation and activation of Treg cell .This kind of inhibitory activity is neither relied direct or indirect contact with the lym -phocytes , nor influenced by inducing immune cells apoptosis .