Objective To construct prokaryotic expression vector carrying Japanese encephalitis virus (JEV) NS1 gene, and to express the vector in E. coli, so to lay a foundation for the further development of JEV early diagnosis kit. Methods The NS1 gene was amplified by RT-PCR, the target gene and prokaryotic expression plasmid pET28a(+) were digested by BamH I and Hind Ⅲ respectively. The target gene was then purified by DNA extraction kit. A 1∶3 molar ratio of vector: insert DNA ligated with T4 DNA ligase to construct the recombinant plasmid pET28a(+)-NS1. The ligated products were transformed into E. coli BL21 (DE3) and the colonies were selected on LB medium with karamycin. After cultivation, positive colonies were picked out and the recombinant plasmid were identified by endonuclease digestions, PCR rand sequencing. The target protein was expressed with induction of IPTG. The expressed proteins mentioned above were then identified and analyzed by SDS-PAGE and Western-blotting respectively. Results The sequencing results of amplified products showed that JEV-NS1 RNA fragments were about 1 300bp in length which were similar as respected. Compared with the published sequence of SA14-14-2 with Blast, the homology of the nucleotide sequence was 100%. The molecular weight of expressed protein was about 45kD, the result of Western blotting proved the specific antigenicity of the protein. Conclusion The specific JEV nonstructural protein 1 is expressed in E. coli successfully and shows high specificity to the antibody. The stable expression of the protein and the analysis of its antigenic specificity provide foundation to develop the early stage diagnosis kit.

BACKGROUND:In the process of cardiac stem cel culture in vitro, the growth microenvironment may have some effects on the cel proliferation.
OBJECTIVE:To investigate the possible mechanism of proliferation and migration of rat cardiac stem cel s cultured in vitro.
METHODS:Cardiac tissues from 10 Sprague-Dawley rats were obtained for primary culture and subculture. Passage 3 cel s were col ected for immunofluorescence staining, and stem cel growth factor receptor (c-kit) and CD45, CD90 were detected. Cultured tissues were col ected and randomly divided into two groups:in group 1, paraformaldehyde fixation, paraffin embedding, hematoxylin-eosin staining, Masson staining, and detecting apoptotic cel s using TUNEL method were conducted;in group 2, EdU labeling of proliferated cel s, immunofluorescent detection of c-kit positive expression, matrix metal oproteinases 2, 9 and transforming growth factorβ1 using immunofluorescent staining were done.
RESULTS AND CONCLUSION:After 7-10 days of myocardial tissue culture, bright and round cel s were visible, and after adhesion, fusiform cel s exhibited strong growth and proliferation ability. Immunofluorescence staining showed a large number of c-kit, CD45 positive cel s but CD90 negative cel s. After culture, a great number of newborn cel s were found, accompanied by apoptosis of myocardial cel s. After EdU staining, the positive cel s were distributed in the myocardial gap, and showed a smal amount of matrix metal oproteinases 2, 9 and transforming growth factorβ1, while in the surrounding myocardium there was a large number of matrix metal oproteinases 2, 9 and transforming growth factorβ1. Taken together, our findings show that cardiac stem cel s could be obtained through myocardial tissue culture in vitro, accompanied by cel proliferation and migration. The mechanism is related to the expression of matrix metal oproteinases 2, 9 and transforming growth factorβ1.

Objective To present the diagnosis and treatment of functional bladder outlet obstruction and to assess the results of transurethral bladder neck incision and alpha-blockers with regard to symptoms and urodynamic findings. Methods From October 1995 to October 2002,39 male patients (age range from 24 to 48 years,with a mean of 37 years) who had dysuria and underwent urodynamic examination,cystourethrography and urethral exploration were diagnosed with functional bladder outlet obstruction.The mean IPSS was 22.5.The mean maximum urinary flow rate was 10.2 ml/s and the mean residual volume was 124 ml.All the patients were treated with transurethral incision of bladder neck and alpha blockers. Results The mean operative duration was 15 min;mean blood loss was 50 ml;mean postoperative hospital stay was 3.5 d.During 1-year follow-up,most of the patients were satisfied with the treatment results.Subjective assessment showed a statistically significant reduction of the voiding complaints.The mean IPSS was 10.1 .The mean maximum urinary flow rate was 22.1 ml/s (range,12.7 to 42.1 ml/s) and the mean residual volume was 49 ml (range,0 to 84 ml). Conclusions Urodynamic examination,voiding cystourethrography and urethral exploration with dilator facilitate the diagnosis of functional bladder outlet obstruction.Treatments with transurethral incision of the bladder neck and alpha-blockers are effective and safe for functional bladder outlet obstruction.

Background The identification of mesenchymal stem cells(MSCs) have provided exciting prospects for cell-based regeneration after myocardic infraction.However cell therapy have inherent limitations such as low survival rate of transplanted cells and insufficient cell number.It is known that cell-matrix adhesion plays a key role in cell proliferation,differentiation and survival,and chemokine CXCL12 may involved in these prcesses.Transfected mesenchymalstem cells with CXCL12 for local secretion of CXCL12 and then explored CXCL12 triggered adhesion of mesenchymal stem cells to extracellular matrix proteins.Mesenchymal stem cells was transfected with CXCL12.?V and ?3 integrins content was evaluated by Western blot analysis.Cell adhesion to extracellular matrix was examined in vitro and cell prolife-ration after transplantation in vivo.Transfection of CXCL12 resulted increased CXCL12 in situ.Increased CXCL12 induced elevated adhesion to fibronectin in vitro and higher survival in vivo.CXCL12 mediated adhesion and proliferation was established by ?V and ?3 integrin subunits.Chemoattractive mechanisms are involved in adhesion processes of mesenchymal stem cells.Increased CXCL12 leads to enhanced expression of ?V and ?3 integrins,which may augment cell survival,proliferation and differrentiation.

Age Factors ; Cardiopulmonary Bypass ; Child ; Child, Preschool ; Humans ; Infant ; Infant, Newborn ; P-Selectin ; blood ; Platelet Glycoprotein GPIb-IX Complex ; analysis

Objective To evaluate hepatectomy for liver metastasis in patients of gastric carcinoma.Methods Clinical data of 32 gastric cancer cases undergoing hepatectomy for hepatic metastatic tumor were reviewed retrospectively from 2006 to 2012.16 cases underwent radical gastrectomy and synchronous hepatectomy for liver metastasis,the remaining 16 cases underwent radical resection of gastric cancer and liver resection heterochronously.The relationship between prognosis and clinicopathology was analyzed.Results The overall survival rates were 84％,50％ and 37％ in 1 year,3 years and 5 years.The median survival time was 32 months.Gastric cancer invasion depth,intravascular tumor thrombi,lymphatic metastasis and intraoperative blood transfusion was related to poor prognosis by single factor analysis,while gastric serosal invasion,tumor thrombus and liver metastasis tumor ＞ 5 cm related to poor prognosis by multiple factors analysis.Conclusions Gastric cancer patients with liver metastasis who underwent hepatic resection can achieve good prognosis if hepatic metastatic tumor ＜ 5cm or the primary gastric cancer does not invade the serosa and without tumor thrombus.

Objective To investigate the application value of CT/MR image fusing in gross tumor volume (GTV) delineation of esophageal squamous cell carcinoma.Methods Twenty-nine patients with esophageal squamous cell carcinoma to be treated with radical surgery underwent routine CT scanning,MR T2-weighted imaging (T2WI) and diffusion weighted imaging (DWI) before surgery.Diffusion-sensitive gradient b values were taken at 400,600,and 800 s/mm2.GTVs were delineated on the CT image,CT/ MR T2WI,and CT/MR DWI respectively.The MR T2W1 image was used as the intermediary for the fusion of the CT image and MR DWI image.The length of GTVs measured under different imaging conditions were compared with the length of the resected specimen of esophagus.Results The GTV length was (44.94 ± 18.46) and (45.05 ±21.47) mm on the CT images and CT/MR T2WI images respectively.When the b values were 400,600,and 800 s/mm2,the esophageal carcinoma GTV length on CT/MR DWI images was (42.12 ± 17.79),(41.18 ± 17.17) and (39.77 ± 17.66) mm,respectively.The coefficient between the esophageal carcinoma GTV lengths on CT/MR DWI images and the pathological lesion lengths was 0.928,0.926 and 0.927 respectively.Conclusions CT/MR DWI images displays esophageal carcinoma GTV length more accurately,thus guiding the delineation of GTV effectively.

Objective This study aimed to evaluate the potential predictive factors for carcinomatous transformation of gastric low-grade intraepithelial neoplasia (LGIEN) on the basis of endoscopic features.Methods The study involved 312 gastric LGIENs that were histologically confirmed by endoscopic forceps biopsy (EFB) between May 2004 and September 2010,and then removed by endoscopic mucosal resection or endoscopic submucosal dissection were enrolled in this study.According to EMR/ESD postoperative pathological findings,they were divided into LGIEN group and the HGIEN group,and compared their endoscopic characteristics.Results There were not significant different between the two group,as the mean age、gender the diameter of the lesions、lesion sites and surface nodularity.The diameter of the lesions was 14.6 ± 8.2 mm in the LGIEN group and 22.0 ± 0.55 mm in the HGIEN group (P ＜ 0.05).42 of 69 gastric adenomas (60.9％)larger than 20 mm in diameter showed HGIEN (P ＜ 0.05).Hyperemia and mucosal ulceration were significant differences in the two groups.Conclusion The LGIEN lesions with these endoscopic characteristics,such as size (＞ 2 cm),surface hyperemia,ulceration should be considered for EMR/ESD.

Objective To investigate the analgesic efficacy and spinal neurotoxicity of intrathecal (IT) different doses of dexmedetomidine in rats. Methods Sixty male SD rats weighing 180-220 g were randomly divided into 5 groups ( n = 12 each): groupnormal control (group C); group IT normal saline (group N); different doses of dexmedetomidine groups received IT dexmedetomidine 0.75, 1.50 and 3.00 μg/kg respectively (groups D1.3). Paw withdrawal threshold to mechanical stimulation (PWMT)with yon Frey filaments and tail flick latency (TFL) to a thermal nociceptive stimulus were measured before (To, baseline) and at 30 or60 rin after IT dexmedetomidine or normal saline administration (T1, T2 ) and the percentage of the maximum possible effect ( MPE ) was calculated. Lumbar segment of the spinal cord ( L4-6 ) was removed for microscopic examination and determination of c-Fos expression (by immuno-histochemistry) at 7, 24 and 48 h after IT dexmedetomidine or normal saline administration. Results PWMT, TFL and the percentage of MPE were significantly increased after IT dexmedetomidine as compared with the baseline values at T0 in groups D1-3 ( P ＜ 0.05). PWMT was significantly higher at T1 and TFL and the percentage of MPE were higher at T2 in groups D1-3 than in groups C and N,and in group D3 than in groups D1,2 ( P ＜ 0.05). At 7,24 h after IT dexmedetomidine c-Fos protein expression was significantly higher in group D3 than in groups C and N( P ＜ 0.05). There was no significant difference in c-Fos expression at 48 h after IT dexmedetomidine between group D3 and groups C and N ( P ＞ 0.05 ). At 24 h after IT dexmedetomidine c-Fos protein expression was significantly higher in group D3 than in other 4 groups( P ＜ 0.05). Slight spinal cord injury was observed at 24 h after IT dexmedetomidine in group D3. Conclusion IT dexmedetomidine has antinociceptive effect. High dose dexmedetomidine IT can produce transient reversible toxicity to the spinal cord.

Objective To study the indications ,surgical techniques of cochlear implantation in bilateral severe sensorineural hearing loss patients with otitis media with effusion .Methods Retrospective study of the data was col-lected from 30 bilateeral severe sensorineural hearing loss patients receiving cochlear implantation .CI was performed with round window insertion in 30 patients with otitis media with effusion .Results One stage operations of CI with round window insertion were carried out for 30 patients with otitis media with effusion .All electrodes were implan-ted successfully ,in which the CI went normally and electrode array were protected well .All implant devices had worked normally and all patients had performed well during an average of 1~3 years follow -up .Conclusion Bilat-eral severe sensorineural hearing loss patients with otitis media with effusion could be performed cochlear implanta-tion with round window insertion in one stage ,and it was safe and effective .