Objective The newly developing gene therapy method and dominant negative mutants were bein g used as new promising HBV therapy method, and a dominant negative mutant of HB V X g ene we have reported in our previous report has some effects both on HBV replica tion and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG 2 2.2.15 cell line in the experiment. To mak e sure the effects of dominant negative mutant of HBx gene, we established a HBx DN stably expressing cell clone, and evaluated the effects of HBx dominant negat ive mutant on HBV gene expression. Methods The prev HBx-GFP dominant mutant and the plasmids pRev Xwt, pRev GFP which contain the wild type X gene or GFP gene then transfected into HepG 2 2.2.15 cells by liposome. The HBsAg, HBeAg by in media were as sayed by RIA and HBV-related RNA were assayed by Northern blot. Results The pRev HBx-GFP, GFP and wild type X constructs can be effectively expressed in HepG 2 2.2.15 cells. The stable expressed HBx -GFP can significantly reduce HBeAg, HBeAg in media and the HBV-related RNA in HepG 2 2.2.15 cells, but not for pRev Xwt and pRev GFP. Conclusions The dominant negative mutant pRev HBx-GFP can significantly inhibit the HBV gen e expression. It also suggested that X gene may be one promising target for HBV gene therapy.

Objective To investigate the clinical significance of changes of serum amylase, CRP and SAA in the diagnosis of acute pancreatitis. Methods The levels of serum and urine amylase, CRP and SAA in patients of mild acute pancreatitis (MAP) and severe acute pancreatitis (SAP) at 24 h, 48 h, 72 h and the seventh day after the onset of pancreatitis were measured. Results The levels of serum, urine amylase, CRP and SAA in SAP patients at 24h were (904.5±402.2)U/L, (2280.3±1207.3)U/L, (155.6±36.2) mg/L, (521.9±109.4)mg/L, respectively, and significantly higher than those of MAP patients (P＜0.05 or P＜0.001). The peak value of serum amylase appeared at 24h, however, the peak value of urine amylase, CRP and SAA appeared at 48 h, and the corresponding values were (2173.5±1110.6) U/L, (185.3±41.4) mg/L and (717.5±144.2)mg/L, respectively. The levels of serum and urine amylase significantly decreases in MAP and SAP patients at the seventh day (P＜0.05). The levels of serum CRP and SAA significantly decreased in MAP patients at the seventh day (P＜0.05), however, the levels of serum CRP and SAA did not significantly decrease in SAP patients at the seventh day (P＞0.05). Serum levels of CRP and SAA were related to the severity of acute pancreatitis. Meanwhile CRP showed a positive correlation with SAA (r = 0.761, P＜0.05). Conclusions The change of serum levels of amylase, CRP and SAA can help early diagnose acute pancreatitis; CRP and SAA may predict the development of SAP at early stage.

Objective To study the development a clinical useful microarray which can detect 4 most commonly and well documented sites for detection of lamivudine-resistance mutants in HBV lamivudine therapy. Methods (1) The selected 16 oligonucleotide probes to screen 4 mutated sites in YMDD region are immoblized in a specific treatment slice by GMS 417 Arrayer. The target 308 bp nucleic acids segment of HBV were amplificated and labeled with Cy5-dCTP fluorescent dye by PCR. After hybridization of target DNA with microarray, the microchips were scanned with Gene TAC LS IV scanner and the data were obtained after processed in computer. (2) To evaluated the specialty and reproducibility of the microarray, the plasmid with Leu515Met, Met539Ile, Met539Val,V542I mutations were condstructed and serve as positive sample. Another 50 sera of lamivudine treated Hepatitis B patients for 6 to 12 months as well as 50 sera without lamivudine administration were assayed by this microarray to evaluated the rate and genetype in lamivudine related resistance mutation. Results The microarray can identify a single base change of selected lamivudine resistance-related mutation and multiple mutation detection by a single assay as well. The specialty are well in agreement with sequencing for 98%. The reproducibility rate of repeat examination is range from 96%-100%. Conclusion The microarray of lamivuine resistance related mutation can identify a single base change as multiple mutations well. And its hight reproducible results may be useful for clinical monitoring lamivudine resistance related HBV mutation.

Objective To detecte the expression of COX-2,VEGF-C and lymphatic vessel density (LVD)in pancreatic cancerous and paracancerous tissues,and investigate their correlation.Methods The expression of COX-2.VEGF-C and LVD in 40 cases of pancreatic cancer tissues and paracancerous tissues and 12 cases of normal pancreas was detected by tissue chip and immunohistochemical assays,and the relationship between them and the cljnicopathological parameters was analyzed. Results The expression of COX-2,VEGF-C in pancreatic cancer tissues were 70.0%(28/40)and 67.5%(27/40),respectively,which were significantly higher than that in paracancerous tissues(42.5%,17/40)and(35.0%,14/40),and that in normal pancreas(8.3%,1/12)and(25.0%,3/12).The LVD in pancreatic cancerous,paracancerous and normal pancreatic tissues were 4.75±2.77,15.2 ±4.70 and 1.67±1.15,respectively.The expression of COX-2 in cancerous tissues and LVD in paracancerous tissues was correlated with tumor differentiation and lymph metastasis;the expression of VEGF-C Was correlated with lymph metastasis.LVD in paracancerous tissues was correlated with the expression of COX-2 and VEGF-C.Conclusions Pancreatic cancer lymphangiogenesis mainly existed in paracancerous tissues,COX-2 and VEGF-C may play an important role in the lymphangiogenesis.

Objective To explore the effect of support with total parenteral nutrition(TPN)or early enteral and parenteral nutrition(EN+PN)on immune function of critically ill neurosurgical patients.Methods In this prospective control study,patients were divided inte TPN group and EN+PN group based on the timing of admission.The changes of immunological indicators including CD3,CD4,CD8,CD4/CD8,CD3/CD25,IgA,IgG,IgM,and serum protein before and after nutritional support were compared.Results The percentage of T lymphocyte subsets CD3,CD4,and CD8,the ratio of CD3+/CD25+,the plasma leveh of IgA,IgM,and IgG,and the serum protein were significantly increased after nutrifional supports(P<0.05,P<0.01).However,compared with the TPN group,the percentages of T lymphocyte subsets(CD3,CD4,and CD8),the ratio of CD4+/CD8+,the plasma levels of IgA,IgM,and IgG,and the serum protein were significantly higher in EN+PN group(P<0.05,P<0.01).Conclusions Both TPN and EN+PN can promote the recovery of immune function,while EN+PN is superior to TPN.Early nutritional support should be provided to critically ill neurosurgical patients.

Objective To investigate the expression of RECK and MMP-9 in pancreatic cancer and to explore the relationship between RECK, MMP-9 expression and the clinicopathological characteristics.Methods PV6000 immunohistochemical method was used to detect the expression of RECK and MMP-9 in 28 cases of pancreatic cancer and 10 cases of normal pancreatic tissue. All the statistical analyses were performed by using SPSS 13.0 statistical software to determine the relationship between RECK, MMP-9 expression and the clinicopathological characteristics. Results The overall positive rate of RECK espression was 46.43% (13/28)in pancreatic cancer, which was significantly lower than that in normal pancreatic tissue (90%, 9/10). The positive rate of RECK espression in Ⅰ + Ⅱ clinical stage (75.0% ,9/12) was significantly higher than that in Ⅲ + Ⅳ stage (25.0%, 4/16 P < 0.05 ). The positive rate of RECK expression in cases without distant metastases (60.0%, 12/60) was significantly higher than that in cases with distant metastasis (12.5%, 1/8,P<0.05). The overall positive rate of MMP-9 was 75% (21/28) in pancreatic cancer, and 20% (2/10) in normal pancreatic tissue. The comparison between these two groups indicated a significant difference (P <0.01 ). The positive rate of MMP-9 in Ⅰ + Ⅱ clinical stage(50.0% ,6/12) was significantly lower than that in Ⅲ + Ⅳ stage (93.8,15/16, P < 0.05). The positive rate of MMP-9 in well differentiation group(33.3%,1/3 ) was significantly lower than that in poor differentiation group ( 100%, 12/12 ,P < 0. 01 ). The expressionof RECK was negatively correlated with the expression of MMP-9 ( r = - 0. 536, P < 0.01 ). Conclusions RECK is lowly expressed in pancreatic cancer, but MMP-9 is highly expressed. RECK and MMP-9 may serve as important markers in the evaluation of tumor stage.

Objective To investigate the apoptosis-inducing effect and anti-proliferative effect of epigallocatechin-3-gallate (EGCG) on human pancreatic cancer cell SW1990 in vitro. Methods The effect of proliferation was evaluated by MTT after the SW1990 cells in vitro were incubated with different concentrations of EGCG (6.25, 12.5, 25, 50, 100 μg/ml). The apoptosis-inducing effect was determined by flow cytometry after the cells were treated with 25 μg/ml of EGCG. The cell cycle of SW1990 cells was detected by flow cytometry after the cells incubated with different concentrations of EGCG (0, 10, 20, 30, 40, 50 μg/ml).Results After SW1990 cell were treated with different concentrations of EGCG (0, 25, 50 μg/ml), the values of A492 were 0.46 ±0.04,0.42 ±0.04,0.27 ±0.03 at 24 h; 0.48 ±0.02, 0.31 ±0.03,0.16 ±0.02at 48 h; 0.51 ±0.01,0.24 ±0.04,0. 14 ±0.04 at 72 h. EGCG inhibited the proliferation of SW1990 in a doseand time-dependant manner(P ＜0.01 ). The apoptotic rates at 24, 48, 72 h were (8.33 ± 1.15 )%, (19.77 ±0.81 )%, (29.17 ± 0.75 )% in the EGCG treatment group; while the corresponding values were (2.77 ±0.45 ) %, (3.20 ± 0.26 ) %, (3.67 ± 0.35 ) % in the control group; and the difference was statistically significant (P ＜0.01 ). After 0, 20, 50 μg/ml of EGCG treatment for 24 h, the percentages of SW1990 cellsin G0/G1 stage were (57.59 ±0.97)%, (62.99 ± 1.91 )%, (68.87 ± 1.88)%, and the percentages of SW1990 cells in G0/G1 stage increased with the increase of concentrations of EGCG, while the percentages of SW1990 cells in G2/M stage decreased with the increase of concentrations of EGCG (P ＜0.01 ). Conclusions EGCG can significantly inhibit the proliferation of SW1990 cells. The mechanism may be related to the apoptosis-inducing effect and the regulation of the cell cycle of the SW1990 cells.

Objective To evaluate the value of the Bedside Index for Severity in Acute Pancreatitis (BISAP) in diagnosing severe acute pancreatitis. Methods Sixty-eight patients with suspected diagnosis of severe acute pancreatitis were collected and were scored by BISAP, APACHE Ⅱ , Ranson and CTSI scoring systems, respectively. BISAP scoring system included the blood urea nitrogen, impaired mental status,systemic inflammatory response syndrome, age, and pleural effusion. The diagnosis criteria of severe acute pancreatitis was BISAP ≥ 3 points or APACHE IⅡ ≥ 8 points, Ranson ≥ 3 points, CTSI ≥ 3 points. The diagnostic accuracy of SAP of these scoring systems was calculated. Results Among these 68 cases, 63.2％(43/68) were graded ≥ 3 points in BISAP scoring system;60.3％ (41/68) were marked ≥8 points in APACHE Ⅱ scoring system; 60.3％ (41/68) were scored ≥ 3 points in Ranson scoring system; and 67.6％(46/68) were scored ≥3 points in CTSI scoring system. There was no statistical difference between BISAP scoring system and other three scoring systems in diagnosing severe acute pancreatitis. Conclusions As a new and simple scoring system, BISAP scoring system can be widely used in the diagnosis of severe acute pancreatitis.

Objective To explore the risk factors,the distribution of etiology and drug resistance status of patients with early infection (3 months) after liver transplantation,and to provide reference for clinical diagnosis and treatment.Methods The clinical data of 112 recipients from February 2014 to December 2015 were collected,and logistic regression analysis was performed on the risk factors of early postoperative infection in liver transplant patients.The independent risk factors of infection after liver transplantation were screened out.At the same time,the results of pathogen culture and drug sensitivity test were statistically described.Results The independent risk factors for infection at 3th month after liver transplantation included the operative time ≥600 min [P =0.003,odds ratio (OR) =9.996,95 ％ confidence interval (95 ％ CI),2.221-44.981],intensive care unit (ICU) ≥6 days (P =0.010,OR =6.306,95％ CI =1.563-25.437),Child-Pugh grade of C (P =0.023,OR =6.298,95％ CI =1.294-30.659).Of the 112 liver transplant recipients,59 had an infection (52.68％),and 168 stains of pathogens were isolated.The positive rate of the specimens was highest in sputum,followed by bile,ascites,drainage and catheter end,blood,deep vein catheter,middle urinary,pleural effusion and peripherally inserted central catheter (PICC).The detectable rate of gram-negative bacteria,gram-positive bacteria,fungi and viruses was 46.43％ (78 strains),29.76％ (50 strains),18.45％ (31 strains),and 5.36％ (9 strains) respectively.Infection occurred mainly within 1 month after surgery,accounting for about 80.36％ (135 strains),especially at 1st week after surgery,accounting for about 34.52％ (58 strains).Gram-positive bacteria had a higher drug resistance rate,including penicillins,macrolides,aminoglycosides,quinolones,linamides,etc.especially in the highest rate of Enterococcus faeciurr.Gram-negative bacteria were individualized based on the different strains of the bacteria,and they were relatively low in the resistance of the carbapene.Conclusion Infection is one of the most common complications after liver transplantation.To reduce the incidence of infection after liver transplantation,efforts should be made to shorten the duration of operation and ICU stay time,improve the basic nutritional status of recipients,and enhance monitoring of the recipient's infection after liver transplantation,to further increase the survival rate of postoperative liver transplantation recipients and improve the quality of life.

Objective To detect serum biomarkers for pancreatic cancer associated diabetes and establish a model for diagnosis.Methods SELDI-TOF-MS was used to detect the differentially expressed serum proteins from 17 pancreatic cancer associated diabetes patients,17 new-onset type Ⅱ diabetes patients and 17 healthy controls,then a model of biomarkers was constructed and validated by Biomarker Patterns Software 5.0.Results Twelve discriminating m/z peaks were identified in the protein fingerprints in 10 pancreatic cancer associated diabetes patients,10 new-onset type Ⅱ diabetes patients and 10 healthy controls.Among them,the three biomarkers of mass/charge ratio 6116,6695 and 8936 were used to construct the model,which could diagnose 90％ pancreatic cancer associated diabetes form control groups.Blind test of other7 samples of three groups showed that 100％ pancreatic cancer associated diabetes,71％ new-onset diabetes and 86％ healthy controls were correctly classified.After searching protein database,there were metallothionein,pancreatic progenitor cell differentiation and proliferation factor-like protein,and fibroblastic growth factor 1,which were close to the weights of the above mentioned 3 differentially expressed proteins.Conclusions SELDI can identify 3 biomarkers for pancreatic cancer associated diabetes and a reliable model for diagnosis of pancreatic cancer associated diabetes is established.