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.

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.

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

Objective To study whether the contrast volume and radiation dose can be reduced by automated contrast injection system(ACIS) in coronary angiography compared with manual contrast injection system(MCIS).Methods 200 patients undergoing coronary angiography with transradial approach in the People′s Hospital of Liaoning Province were enrolled in the study from January 2016 to June 2016.They were divided into the ACIS group (n=100) and the MCIS group (n=100).The clinical data, the net amount of contrast the total amount of contrast media consumed, number of angiographic views performed, fluoroscopy time, air kerma (AK) and dose area product (DAP) of the two groups were statistically analyzed.Results There were no statistical differences in the clinical data, the net amount of contrast used, number of angiographic views performed and fluoroscopy time between the two groups (all P>0.05).The total amount of contrast media used, AK, and DAP were less in the ACIS group than in the MCIS group (all P<0.05).Conclusions The volume of contrast consumption and radiation dose can be reduced by ACIS during coronary angiography with transradial approach compared to MCIS.

Objective To study the effects of high albumin, high glucose and low bovine serum on the phenotypic transformation of renal tubular epithelial cells. Methods The normal human kidney proximal tubular eel] line (HKC) was cultured for 30 days in the presence of high albumin (1.5 g/L), high glucose(25 mmol/L) and low bovine serum(2% ) . Morphological changes were observed by electronic microscopy. Immunohistochemistry stain was used to examine the expression of cytokeratin, vimentin, a-SMA, collagen Ⅰ and TGF-pl protein. Western blot was applied to further detect the process of collagen I protein expression, and in situ hybridization was used to examined the expression of collagen Ⅰ gene. Results Renal tubular epithelial cells cultured in high albumin, high glucose and low bovine serum showed obvious morphologic changes, including elongated shape, decrease of microvilli and mitochondria, and increase of rough endoplasmic reticulum under electronic microscopy. Immunohistochemistry stain revealed the reduction of cytokeratin, and enhancement of vimentin, ?-SMA, TGF-?1 and collagen Ⅰ. Western blot demonstrated that the expression of collagen Ⅰincreased in a time-dependent manner, and in situ hybridization showed that collagen type Ⅰ mRNA increased as well. Conclusion High albumin, high glucose and low bovine serum induce phenotypic transformation of renal tubular epithelial cells into mesanchymal cells.

Objective: To investigate expression of TRAP in human tumors and correlativity between TRAP and hTERT and to explore the role of TRAP in tumorigenesis. Methods: TRAP was expressed in E.coli . and polyclonal antibody was prepared by immunization of New Zealand rabbits. Expressions of TRAP gene and hTERT in human tumors were analyzed by immunohistochemistry. The eukaryotic expression vectors of TRAP were constructed and transferred stably into HeLa cells to be analysed with Northern blot, TRAP PCR ELISA, growth curve, nude mouse transplantation. Results: Polyclonal antibody was obtained from New Zealand rabbits immunized with TRAP fuse protein, which was inducibly expressed in E.coli ., and its specificity was verified in Western blot. TRAP staining was detected in 213 (86.2%)of 247 malignant tumors. On the contrary, TRAP in all tissues adjacent to tumors was negative.There is a obvious difference between borderline,pre cancerous and benign lesion in the expression of TRAP.Furthermore, hTERT staining was conducted and its result was consistent with that of TRAP. Up regulation and down regulation of hTERT expression and telomerase activity were observed respectively in HeLa cells transfected stably with sense and antisence TRAP recombination. Increased proliferation presented in the former and growth inhibition in the latter. Besides, in nude mice, tumorigenecity of sense TRAP transfected HeLa cells was enhanced while tumor growth of antisense TRAP transfected HeLa cells was retarded. Conclusion: The expression of TRAP, which could be detected in most of human cancers and part of pre cancerous lesions, was correlated with expression of hTERT. The status of TRAP expression could regulate telomerase activity and present effects on growth and tumorigeneity of cancer cells. This suggests that TRAP is involved in human tumorigenesis through regulation of telermarase activity.

Objective To investigate the expression of HLA-G in lymphocytes,CD3+ T cells,and CD14+ monocytes from patients with rheumatoid arthritis (RA) and their associations with disease activity,disease duration,medication used and clinical parameters.Methods We studied 68 RA patients as well as 63 healthy controls.HLA-G expression was analyzed by flow cytometry in peripheral blood mononuclear cells (PBMCs).Mann Whitney U tests and Spearman rank correlation coefficient test were used for statistical analysis.Results We found that lymphocytes as well as CD3+ T cells,and CD14+ monocytes in RA patients showed a diminished expression of HLA-G compared with healthy controls [1.98％(1.17％-3.66％) vs 2.93％ (1.70％-5.40％),U=1624.5,P＜0.05; 1.35％(0.57％-2.79％) vs 2.16％(1.15％-4.45％),U=1387,P=0.000; 22.65％ (10.59％-36.81％) vs 32.76％ (20.73％-44.62％),U=1526,P＜0.01].Spearman analysis showed that the levels of HLA-G+ lymphocytes as well as CD3+ HLA-G+ T cells was negatively associated with DAS28 score(r=-0.288,P=0.017; r=-0.373,P=0.001),but no significant correlation was detected between the levels of CD14+HLA-G+monocytes and DAS 28 score (P＞0.05).In addition,the levels of expression of HLA-G was not correlated to disease duration,medication used,ESR,CRP,RF,anti-CCP and ANA (P＞0.05).Conclusion Decreased expression of HLA-G are detected in lymphocytes,CD3+ T cells,and CD14+ monocytes from pa-tients with RA,and the abnormality of HLA-G may play an important role in the development of RA.

Objective To analyze the viral infection in patients with acute exacerbation of chronic obstructive pulmonary disease(COPD)and its clinical significance.Methods 350 patients with acute exacerbation of COPD were collected.The viral infection and clinical manifestations,pulmonary function and imaging features were detected and analyzed.Results A total of 104 patients with viral infection,the proportion was 29.7%.Single virus infection in 74 cases(71.2%),the highest proportion was FluB(19.2%)in pathogen type,followed by FluA(15.4%).Multiple virus infection in 30 cases(28.8%),the highest proportion was RSV+FluB(11.5%).The virus infection in patients with sore throat(20.2% vs.6.9%),lung function grade(32.7% vs.20.3%),presence of pulmonary inflammatory lesions(57.7% vs.40.7)and the death rate(12.5% vs.0.8%)were significantly higher than those of the combined group of virus infection,the proportion of lung function was significantly lower than patients without viral infection,the differences were statistically significant(x2=6.546,5.477,4.987,4.124,3.997,all P<0.05).The proportion of respiratory failure in patients with acute COPD infection was 86.5%,which was higher than 62.6% of patients without viral infection,the difference was statistically significant(x2=19.837,P<0.05).Conclusion Acute exacerbation of COPD has a high proportion of virus infection,and the prognosis of patients is poor.

Objective To study effects of multimodal analgesia on postoperative delirium (POD)in elder patients with hip fracture.Methods One hundred and eight elder patients with hip fractures were gathered in a prospective study.Fifty-nine cases were included in the group of multimodal analgesia (25 males,34 females),with an average age of 72.91±5.42 years,and 35 were treated with hip replacements,the other 24 were internal fixations.Forty-nine cases were in the group of conventional analgesia (22 males,27 females),with an average age of 72.14±4.93 years,29 were treated with hip replacements,20 were internal fixations.Assessments of delirium were based on confusion assessment method.Local infiltrated anesthesia,patient control intravenous analgesia,and intravenous nonsteroid anti-inflammatory drug were applied in the group of multimodal analgesia.For the other group,morphine would be given only when patient complained pain or there was POD.Postoperatively,visual analogue scale (VAS),onset of delirium,other correlative data were recorded by an independent researcher.All patients underwent POD were managed with intensive pain management,and then reevaluated.Results VAS in multimodal analgesia group [Day1:2.10±1.43(resting),4.74±1.45 (active) and Day 3:1.01±0.92 (resting),3.31±1.36 (active)] were significandy lower than that in the other group [Day1:4.67±1.33 (resting),7.44±1.59 (active)and Day 3:2.24±1.39 (resting),5.06±1.46 (active)].PODs were detected in 15 (30.6％) in group of conventional analgesia; while in group of multimodal analgesia,there were 7 (11.9％).All POD were given intensive pain managements by injection of 10 mg morphine and achieved relief of deliriums.Conclusion Postoperative multimodal analgesia may reduce the incidence of POD.First aid of intensive pain management may help to control POD.

ObjectiveTo investigate the application of dexmedetomidine attenuating hemodynamic fluctuation in patients with cerebral aneurysm during anesthesia induction.Methods Sixty patients undergoing elective interventional procedure with cerebral aneurysm were divided into low dose of fentanyl group (group LF),high dose of fentanyl group (group HF),and low dose of fentanyl and dexmedetomidine combination group(group FD) by random digits table method with 20 cases each.Before induction of anesthesia,the patients in group FD received dexmedetomidine 1 μ g/kg for 10 minutes,and the others received 0.9％ sodium chloride with the same volume.During anesthesia induction,fentanyl 3 μ g/kg in group LF and group FD,and fentanyl 5 μ g/kg in group HF.Other anesthetics were equalized.Systolic blood pressure (SBP),diastolic blood pressure (DBP) and heart rate (HR) were monitored and recorded at 3 min utes after lying on table (T0),before intubation (T1),immediately and 3 minutes after intubation (T2,T3).The differences between the maximum and the minimum of SBP,DBP and HR were calculated in these time points as fluctuation values,named as △SBP,△DBP and △HR.Results△SBP,△DBP and △HR in group FD [ (26.9 ± 14.8) mm Hg( 1 mm Hg =0.133 kPa),(10.7 ± 8.9) mm Hg,(12.5 ± 4.3 ) times/min ]were lower than those in group LF [ (40.4 ± 15.6) mm Hg,(20.3 ± 9.4) mm Hg,(30.1 ± 15.0) times/min ](P ＜ 0.05 ),as well as △SBP and △HR in group HF [ (29.8 ± 16.8 ) mm Hg,( 19.5 ± 7.4) times/min ]were lower than those in group LF(P ＜ 0.05).While △HR in group FD was lower than that in group HF (P ＜0.05).The usage of atropin in three group had no statistical significance during anesthesia induction (P =0.364),but more ephedrine was used in group HF than in group LF [30％(6/20) vs.5％(1/20),P=0.032 ].Conclusion Dexmedetomidine 1μ g/kg injected before anesthesia induction,which could prevent intubation reaction,blood pressure serious falling after intubation,and provide more stable hemodynamics,is particularly applicable for anesthesia induction in patients with cerebral aneurysm.