Objective To investigate the feasibility of transfecting gene into HepG2 by therapeutic ultrasound combined with microbubble,and to explore the optimal ultrasonic parameters for high transfection efficiency.Methods HepG2 cells were cross-interval planted in 24-well plates.EGFP plasmid DNA and microbubbles were added to the cultured HepG2 and three parameters of the therapeutic ultrasound were optimized.Firstly,set ultrasonic intensity gradient (group A,from A1 to A5),which were 0.4 W/cm,0.8 W/cm2,1.2 W/cm2,1.6 W/cm2 and 2.0 W/cm2 respectively.Secondly,set the duty cycle gradient group (group B,from B1 to B3),which were 10％,20％ and 50％ respectively.Lastly,set the exposure time group(group C,from C1 to C3),which were 30 s,90 s and 180 s.After 24 hours,the expression of GFP was observed by fluorescence microscopy,the transfection rates was detected by flow cytometry,the cell death were assessed by trypan blue exclusion.Results The plasmid transfection efficiency was varied under different ultrasonic parameters.Within a certain range,the increasing of parameters can improve the transfection efficiency,but when the parameters were too large (eg.the intensity＞1.6 W/cm2 or 50％ duty cycle),the actual transfection efficiency decreased due to the increased cell death.When the ultrasonic intensity was 1.2 W/cm2,the transfection efficiency and mortality were better than the other subgroups of group A.When the duty cycle was 20％,the transfection efficiency and mortality rate were better than the other subgroups of group B.Continue to increase the exposure time over 90 s was not statistically significant (P ＞0.05) for transfection efficiency and cell mortality.Conclusions The ultrasound-targeted microbubble destruction is an efficient method for gene delivery in vitro.The transfection efficiency vary greatly under different parameters,so optimization parameters is conducive to the promotion of gene transfection.

Objective To investigate the effects of transplantated tumor capillary permeability under microbubble enhanced diagnostic ultrasound exposure.Methods Fourty eight rats were randomly divided into four groups after Walker 256 carcinomas were subcutaneousely implanted,named the control group (A),microbubbles group (B),ultrasound group (C),microbubbles and ultrasound group (D).Using Evans blue(EB) as the indicator for tumor capillary permeability,the microbubbles were injected through the vein with diagnostic ultrasound frequency as 1.75 MHz,mechanical index as 1.4.The spillover of EB was estimated under confocal laser microscope,meanwhile the contents of EB in the tumor tissues were calculated according to the standard curve and spectrophotometry.Results EB spillover was observed around the capillaries in the group D,while EB spillover was only observable on capillary in group A,B and C.The EB content was also significantly higher than those in group A,B and C.Conclusions Exposure to both diagnostic ultrasound and microbubbles results in increased capillary permeability of rat tumor tissues,which might become a potential therapeutic method on clinical chemotherapy.

Objective To compare bolus infusion and replenishment using real low mechanical index contrast enhanced ultrasound in assessing the change of renal cortical perfusion.Methods Using dopamine (i.v.) at the dose of 0.5,2.0,5.0μg · kg- 1 · min- 1 to change renal blood perfusion of 20 rabbits,then during bolus or contant injection of SonoVue,at coded pulse inversion mode,real-time contrast ultrasound was performed,the latter method needed destroying microbubble at a high MI when amplitude reach a steady state,then recording the replenishment,peak intensity(A) and time to peak (PPT) were obtained through raw time-intensity curve,and slope rate of TIC(k) was acquired by curve fitting,standard effective renal plasma flow(ERPF) was measured through 4-aminohippuric acid clearancerate method,meanwhile correlations between ERPF and parameters were analyzed,as well as the paired samples t test for each parameter before and after dopamine administration.Results The ascending branchs of raw TIC of bolus infusion increased sharply and were approximately straight,then descended gradually,while that of replenishment looked like two straightlines with different slopes,then stayed horizontal Both the value of A of two methods were positively correlated with ERPF ( r b =0.85,r re =0.66),and were different at the same ERPF,meanwhile the value of TTP were negatively correlated with ERPF( r b =-0.92,r re =- 0.76),and there were no statistically difference between the two methods.k from Gamma fitting was far from correct,while k from exponential fitting was apparently correlated with ERPF ( r re =0.77 ).Conclusions Both bolus injection and constant injection-replenishment method can assess renal cortical blood perfusion,TIC parameters A and TTP represent regional blood volume fraction and microbubble velocity respectively.Bolus-infusion with real low mechanical index is more precise and available.Comparing with k,TTP is more appropriate to reflect perfusion velocity.

Objective To construct and identify an eukaryotic expression plasmid containing rat hepatocyte growth factor(rHGF)gene and rat augmenter of liver regeneration(rALR)gene,so that to provide experimental basis for developing new treatments of hepatic fibrosis.Methods The gene fragments of rHGF and rALR were amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR by polymerase chain reaction(PCR),respectively,then were spliced by overlap extension PCR with a linker,and the fusion gene rHGF-linker-rALR was constructed.The fusion gene was directionally inserted into the eukaryotic expression plasmid pcDNA3.1 between restriction sites of Kpn Ⅰ and Xba Ⅰ to construct the recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR,and the new constructed recombinant plasmid was identified by double restriction digestion and DNA sequencing.Results DNA fragments of 2200 bp and 400 bp were observed after the electrophoresis of products amplified from recombinant prokaryotic plasmid pUC18-rHGF and pUC18-rALR,respectively,which was consistent with the theoretical value.The electrophoresis of fusion gene rHGF-linker-rALR obtained by overlap extension PCR technique showed only a 2 600 bp DNA fragment,which was in accordance with the expected value.Electrophoresis of products of pcDNA3.1-rHGF-linker-rALR digested with Kpn Ⅰ and Xba Ⅰ showed two DNA fragments with 2600 bp and 5400 bp,which were both consistent with the expected value.The sequences were confirmed correctly by DNA sequencing.Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1-rHGF-linker-rALR is successfully constructed,which provides experimental basis for developing gene therapy of hepatic fibrosis.

ObjectiveTo investigate the clinical value of endoscopic esophageal submucosal tunnel resection of gastric fundus-cardiac tumors originating from muscularis propria.Methods Clinical date of 18 patients with gastric fundus-cardiac submucosal tumors originating from muscularis propria who underwent endoscopic esophageal submucosal tunnel resection from January 2011 to December 2011 were retrospectively collected and analyzed.ResultsAll lesions were successfully and completely resected in 18 patients,with sizes ranging from 0.7 cm to 7.2 cm,mean (2.43 ± 1.91 ) cm.Pneumoretroperitoneum,pneumomediastinum and pneumohypoderma occured during the procedure in 2 cases,but spontaneously resolved in 3 days.Fever with increased WBC within 24 h after the procedure occurred in one patient,and was cured in two days with antibiotics.There were no severe complications including bleeding,perforation or death.All patients could have liquid diet 3 days later after the operation.Follow-up endoscopy at 1 week after the operation showed a healing of esophageal incision.ConclusionEndoscopic esophageal submucosal tunnel resection is a safe and effective method for gastric fundus-cardiac submucosal tumors originating from the muscularis propria,lessening the difficulty of traditional endoscopic resection.

ObjectiveTo study the diagnostic value of double balloon endoscopy (DBE) for gastrointestinal disease.MethodsClinical data of 53 patients with suspected intermediate gastrointestinal diseases who underwent DBE procedures were retrospectively analyzed for detection rate,complications and the incidence of adverse reactions.ResultsAll of 53 patients underwent successful DBE,Procedures were performed via mouth ( n =26),anus ( n =9 ) and the combined routes ( n =18 ).And definite diagnosis was made in 46 cases (86.79％),including upper gastrointestinal (above ligament of Treitz) disease in 3 cases (5.66％),intermediate gastrointestinal disease in 41 cases (77.36％ ) and lower gastrointestinal disease in 2 cases (3.77％).All 53 patients were tolerant to the operation and anesthesia.No severe complications like bleeding,perforation,intestinal torsion,mesenteric tear,pancreatitis or anesthetic accident occured.Several patients showed abdominal flatulence,transient abdominal pain.Some patients in oral route reported slight throat discomfort or slight nasal bleeding.Incarceration occurred in 1 case due to friction of endotracheal intubation and the silicone tube,which was released after deflation and removed out.2 patients showed mild diarrhea with low fever,and the symptoms were self-healing without special treatment in 48 h.ConclusionDBE is of high diagnostic value for gastrointestinal diseases with a high safety.

Objective To investigate the clinical value of endoscopic biliary stent drainage for obstructive jaundice due to hepatic portal metastatic carcinoma. Methods Patients with obstructive jaundice arising from hepatic portal metastatic carcinoma were selected and treated with consent by plastic endoscopic bile duct stents since 2006. Success rate and survival were evaluated. A total of 38 patients with obstructive jaundice were treated and analyzed. Hepatic portal metastatic carcinoma included 13 cases of liver cancer, 3gallbladder carcinoma, 14 gastric carcinoma, 2 esophageal carcinoma, 1 ileum adenocarcinoma and 5 pancreatic carcinoma. Results Stents were successfully placed in all patients and jaundice rapidly subsided after the endoscopic procedure. Follow-up life span was 92-521 days, mean 264. 42 ± 104. 41 days. During follow-up, biliary stents were replaced in 5 patients in 3-14 months ( mean 8. 6 ± 4. 1 months) because of stent displacment in 1 case, biliary stone obstruction in 2 cases and carcinomatous obstruction in 2 others.Conclusion For patients with obstructive jaundice arising from hepatic portal metastatic carcinoma, endoscopic biliary stent drainage is effective and can prolong life span to some degree.

0.05). The blood glucose, glycosylated hemoglobin and oxidized low density lipoprotein degrade, insulin resistance were improved in probucol group after treatment, while the adiponectin level was increased(P

Objective To observe the clinical effects of external application of Chinese medicine ion introduction through acupiont in the prevention and treatment of hyperamylasemia and pancreatitis following endoscopic retrograde cholangio pancreatography (ERCP). Methods Three hundred subjects were divided into treatment group and control group randomly, and treated with routine therapy after surgery, moreover, treatment group was treated with external application of Chinese medicine ion introduction through acupiont. The incidence of hyperamylasemia and pancreatitis, the time of serum amylase returned to normal in patients with hyperamylasemia and pancreatitis was observed, and the abdominal pain after ERCP was scored. Results Excluding 17 cases according to the exclusion criteria, the treatment group included 142 cases and the control group included 141 cases. The hyperamylasemia and pancreatitis happened less frequently in the treatment group than in control group, but there was no significant difference (P>0.05). The time of serum amylase returned to normal in patients with pancreatitis was (4.25±0.95)d in treatment group and (5.28±1.11)d in control group, with no significant difference (P>0.05). The time of serum amylase returned to normal in patients with hyperamylasemia was (2.88 ± 0.78)d in treatment group and (3.81±1.62)d in control group, showed a significant difference (P<0.05). The score of abdominal pain was 0.95±1.04 in treatment group and 1.21±1.12 in control group, showed a significant difference (P<0.05). Conclusion External application of Chinese medicine ion introduction through acupoint can promote the recovery of the patients postoperative ERCP combined with hyperamylasemia or pancreatitis, and can better alleviate abdominal pain.

Objective To investigate the role of endoscopic retrograde cholangiopancreatography (ERCP) in the diagnosis and treatment of adult biliary ascariasis.Methods Clinical data of 19 cases of adult biliary ascariasis diagnosed and treated with ERCP during Jan 2005 to Dec 2012 were retrospectively analyzed.Results Among the 19 adult patients with biliary ascariasis,11 were male,and the mean age was (62.7± 10.8) years.Eight cases lived in the countryside,7 cases in the integration of urban and rural area,and 4 cases in urban area.All cases had histories of eating raw fruits and vegetables.Living worms were found in 9 cases.Four cases were complicated with common bile duct stones.Petrification of residual dead worms in the common bile duct was found in 10 cases.All ascariasis and residual bodies were removed successfully by ERCP.There were no postoperative complications except for 3 cases of hyperamylasemia.Conclusions Adult biliary ascariasis commonly occurs in the elderly,and is frequently complicated with common bile duct stones.ERCP is efficacious in treating biliary ascariasis.