This study aimed to construct full-length cDNA clones of the Japanese encephalitis virus (JEV). SA14-14-2 strain and discuss the feasibility of constructing chimeric viruses for exogenous gene expression based on the JEV genetic skeleton. Long-fragment RT-PCR techniques were applied to amplify JEV cD-NAs, and two amplified fragments with corresponding restriction endonuclease sites at both ends were cloned into the pACYC184 vector sequentially. Using standard molecular techniques, the enhanced green fluorescent protein (EGFP) gene was inserted into the 3' non-coding region of JEV as a reporter gene. After in vitro transcription and transfection procedures, wild-type JEV and chimeric JEV that expressed the EGFP as the reporter gene were successfully rescued. The recovered viruses were characterized by RT-PCR, plaque assays, and direct fluorescence microscopy. After six serial passage generations, the stability of the recovered viruses were studied in terms of virus growth characteristics and structural gene expression. The results showed that cDNA clones of rJEV and rJEV-EGFP were successfully constructed and rescued in BHK-21 cells after in vitro transcription and transfection. Each generation of the recovered viruses was stable and the chimeric virus rJEV-EGFP could stably express EGFP. The findings of this study indicate that both rJEV and rJEV-EGFP could be constructed and rescued in BHK-21 cells, and the JEV SA14-14-2 strain could be obtained as a viral vector to express foreign genes.
Cloning, Molecular ; DNA, Complementary ; genetics ; metabolism ; Encephalitis Virus, Japanese ; genetics ; metabolism ; Encephalitis, Japanese ; virology ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Vaccines, Attenuated ; genetics ; metabolism ; Viral Vaccines ; genetics ; metabolism

Objective To analyze the characteristics of cardiovascular two-dimensional (2-D) and Color Doppler echocardiography for patients with Marfan's syndrome. Method 23 cases of Marfan's syndrome were examined by transthoracic 2-D and Color Doppler echocardiography.Results Aneurysms of the ascending aorta and aortic sinusal were found in all of these cases. Aortic regurgitation and left ventricular enlargement coexisted with all of these cases. Fourteen cases complicated with type A and three with type B aortic dissection. Conclusions 2-D and Color Doppler echocardiography is a rapid non-invasive,reproducible, comprehensive medical imaging technique and can be regarded as an initial imaging model to screen and diagnosis of Marfan's syndrome.

Objective To discuss the clinical effects of anal cushion suspensory and external hemorrhoid airfoil-shaped suture in treatment of ring-ship mixed hemorrhoid,improve the level of the surgical treatment of ring-ship mixed hemorrhoid.Methods One hundred and twenty patients with ring-ship mixed hemorrhoid were divided into treatment group (60 cases treated by anal cushion suspensory and external hemorrhoid airfoil-shaped suture) and control group (60 cases treated by Milligan-Morgan operation) according to the operation method.The postoperative pain,hospitalization time,wound healing time,postoperative complications,etc were observed and compared.Results The cure rate was 100.0％ (60/60) in treatment group and 70.0％ (42/60) in control group,and there was significant difference between two groups (P ＜ 0.05).The hospitalization time,wound healing time was (8.58 ± 0.89),(14.00 ± 0.72) d in treatment group,(10.20 ± 0.30),(19.00 ± 0.21) d in control group,and there was significant difference between two groups (P ＜ 0.05).The incidence of postoperative complications and the degree of postoperative pain in treatment group was lower than that in control group,and there was significant difference between two groups (P ＜ 0.05).Conclusion Anal cushion suspensory and external hemorrhoid airfoil-shaped suture in treatment of ring-ship mixed hemorrhoid has advantage of shorter wound healing time,less postoperative pain,less postoperative complications.

Objective To describe the characteristic endoscopic ultrasonography (EUS) features of gastric ectopic pancreas.Methods Totally 23 patients were diagnosed pathologically as having gastric ectopic pancreas.The EUS images of the lesions were retrospectively reviewed regarding layer of origin,size,growth pattern,margin,and internal echo pattern.Results The coincidence of EUS and pathologic diagnosis was 91%(21/23).EUS revealed that the lesions originated from the second,third,and/or fourth layers of the gastric wall.Most lesions were heterogenous,mainly isoechoic.The borders of the lesions were indistinct in 70%(16/23) patients. Anechoic cystic or tubular structures within the lesions appeared in 10 of the 23 lesions(43%).The mean longest/shortest diameter ratio of ectopic pancreas was 1.7.Conclusions Characteristic EUS features of gastric ectopic pancreases include:mural lateral growth pattern,third layer (submucosa) origin,indistinct margin,intermediate echogenecity,anechoic areas,and fourth-layer thickening.Careful assessment of the EUS findings may be a useful aid in the diagnosis of gastric ectopic pancreas.

Objective To evaluate the efficacy of narrow-band imaging (NBI) in diagnosis of color-ectal neoplasm. Methods A total of 96 colorectal neoplasm from 78 patients were observed under NBI, and the findings were compared with those from magnifying chromo-endoscopy and pathologic examinations. Re-suits The sensitivity of conventional colonoscopy in diagnosis of polyps was 78. 7%, which was increased to 99% (P < 0. 05) under NBI, with the clear identification of the shape and boundary of the lesions. Magnif-ying NBI colonoscopy provided images of pit patterns similar to those from chromo-endoscopy in diagnosis of type Ⅱ , ⅢL, Ⅳ and ⅤN, which showed better recognition than conventional colonoscopy, but less sensitiv-ity than chromo-endoscopy. Superficial vascular morphologic features could be better classified with NBI, with sensitivity of 100% and specificity at 87. 8%, which was superior to conventional colonoscopy but infe-rior to chromo-endoscopy. Conclusion Both chromo-endoscopy and NBI colonoscopy have better sensitivity and specificity in detection of colon polyps, while chromo-endoscopy reveals clear superficial structure of le-sion and pit pattern, and NBI demenstrates capillary morphology, which can distinguish neoplasm from non-neoplasm colorectal lesions. NBI, easy and convenient to switch, is an effective technique to make early di-agnosis of colorectal neoplasm.

Objective To investigate protective activity against Aβ25-35-induced cytotoxicity in PC12 cells of different extracts and ursolic acid, which were isolated from pyrola decorata. Methods Aβ25-35-induced cytotoxicity in PC12 cells was established as the model in vitro. The cultured PC12 cells were divided into blank control group, DMSO control group, model control group, different extract groups of pyrola decorate and ursolic acid(UA) group. The different extract groups included ether extract (PE), acetidin extract (AE), n-butanol extract (BE), the water extract (WE), 50% ethanol extract (HEE). MTT assay was used to test the optimum concentration, and the number of viable cells in culture medium was measured by ELISA at 490 nm wavelength. Results The cell viabilities in different extracts groups(PE, AE, BE, WE, HEE) were respectively 89.3%, 77.2%, 79. 2%, 75. 1% ,74. 0% at the concentration of 5. 0 mg ? mL-1 . Moreover, ursolic acid showed the best neuroprotective activity (88.9%) at the concentration of 500 μg?L-1 . Compared with model control group, the survival rate of each group was remarkably increased, and the protective activities of PE and UA were more significant among them. Conclusion Different polar extracts of pyrola decorata and isolated ursolic acid have neuroprotective effects on Aβ25-35-induced cytotoxicity in PC12 cells in certain degrees.

Objective To produce a kind of biliary stent with Mitomycin C-eliting and evaluate the availability and safety in biliary benign stricture model of rabbit.Methods 36 New Zealand rabbits were fulgurize choledochus to establish model of biliary benign stricture.Rabbits were randomly divided into Mitomycin C-eluting stent group 1 (n =12),polyurethane stent group 2 (n =12) and control group 3 (n =12) one month later.General conditions,survival of the animals and changes in liver function were observed after surgery.The histological changes of bile duct were observed after 30 days.The immunohistochemistry SP method was used to measure transforming growth factor-β1 (TGF-β1) and α-Smooth muscle actin (α-SMA) expression.Results Stricture was improved in the two stent groups.In Mitomycin C-Eluting stent group total bilirubin dropped from 5.56 μmol/l to 0.82 μmol/l,and in polyurethane stent group total bilirubin dropped from 6.72 μmol/1 to 0.87 μmol/l.The total bilirubin decreased in both two stent groups but no statistically significant between the two stent groups,and there were no improvement in control group.Diameter of the stricture bile duct in group 1 was expanded bigger than in group 2 according to histology observation.Inflammatory cell infiltration and collagen fibroplasia in the submucosal were obviously observed in control group.The immunohistochemistry results showed that the TGF-β1 and α-SMA strongly expressed in the stenosis bile duct of group 3.The expressions in group 2 were lower than group 3,but higher than in group 1.And there was significant difference between the two stent groups (P ＜ 0.05).Conclusion The new Mitomycin C-Eluting stent is safe and provides enhanced local drug delivery.It also can inhibit the form of Biliary scar to a certain degree.

Objective To investigate the efficacy of pancreatic duct guidewire pre-occupying in ERCP with difficult biliary cannulation.Methods During a four-year study period from June 2008 to June 2012,a total of 3505 patients were included in this retrospective analysis.Initial biliary cannulation method consisted of single-guidewire technique for up to 5 attempts,followed by double-guidewire technique when repeated unintentional pancreatic duct cannulation had taken place.Pre-cut papillotomy technique was reserved for when double-guidewire technique had failed or no pancreatic duct cannulation had been previously achieved.Biliary cannulation success and post-ERCP complication rate were compared.Results Single-guidewire technique was characterized by statistically significant higher success rate (93.4％),compared with the double-guidewire technique (54.8％,P ＜0.001),pre-cut failed double-guidewire technique (81.3％,P ＜0.001) or precut as first step method (84.6％,P =0.011).Pre-cut failed double-guidewire technique and pre-cut as first step method offered a statistically significantly more favorable outcome compared with the double-guidewire technique (both P ＜ 0.001).The incidence of post-ERCP pancreatitis did not differ in a statistically significant manner among the four methods.Numbers of patients who got bleeding in pre-cut papillotomy technique and sphincterotomy after successful single-guidewire technique were 5 and 2 respectively.One case of perforation was recorded using pre-cut papillotomy technique.There was no procedure-related mortality within 30 days.Conclusion Although double-guidewire technique success rate proved not to be superior to singleguidewire technique or pre-cut papillotomy,it is considered highly satisfactory in terms of safety in order to avoid the risk of a pre-cut when biliary therapy is necessary in difficult-to-cannulate cases.

Objective To investigate the effect and mechanism of decitabine (DAC) on the proliferation of human cholangiocarcinoma CCLP1 cells in vitro and in vivo.Methods After treated with various concentrations of DAC,cell growth inhibition rates were determined by CCK-8 assay.Cell cycle and cell apoptosis were analyzed by flow cytometry.Cell autophagy was observed under fluorescence microscope.The effect of DAC on the growth of cholangiocarcinoma in vivo was determined in a CCLP1 mice xenograft model.Results The proliferation rate of CCLP1 cells in the DAC-treated group decreased in a time-concentrated dependent manner.After treatment with DAC,the cell cycle of CCLP1 cells was arrested at the G2/M phase.The apoptosis rate was significantly higher in the treatment group over the control group.Cell autophagy was observed after treatment with DAC in CCLP1 cells.The tumor growth of implanted CCLP1 cells significantly slowed down after the mice were treated with 0.8 mg/kg DAC,6 times weekly for 2 weeks.Conclusion DAC can inhibit the proliferation of cholangiocarcinoma in vitro and in vivo.

Objective To investigate the clinical value of KL-6 mucin in differential diagnosis of benign and malignant obstructive jaundice.Methods A total of 41 patients with malignant tumors and 15 with benign lesions were recruited in the study.KL-6 mucin levels of bile and serum were measured using enzyme-linked immunosorbent assay (ELISA).The serum alpha-fetoprotein (AFP),carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 levels were also measured using radioimmunoassay (RIA).All data were analyzed by Mann-Whitney U test and Receiver Operating Characteristics (ROC)curves.Results CEA,CA19-9,and KL-6 mucin in serum and KL-6 mucin in bile in patients with malignant tumors were significantly higher than those in patients with benign lesions (P ＜ 0.001).There were no significant differences in serum AFP between the two groups.An receiver operating characteristic (ROC)curves analysis revealed that the detection of KL-6 mucin in bile was reasonably accurate in differentiating malignant obstructive jaundice from benign (area under curve [AUC] 0.954).While the AUC of the ROC curves for serum AFP,CEA,CA19-9,KL-6 mucin and KL-6 in bile and 0.954 were 0.657,0.753,0.894,0.846 and 0.954 respectively.Using the ideal threshold as the cut-off point,the sensitivity and specificity of KL-6 mucin for differential diagnosis of benign and malignant obstructive jaundice was 90.24％and 93.33％.Conclusion As a tumor marker,KL-6 mucin in bile is valuable in differential diagnosis of benign and malignant obstructive jaundice.