Objective To construct recombinant full length genotype B and C hepatitis B virus (HBV)and to examine HBV DNA replication and hepatitis B surface antigen(HBsAg),hepatitis B e antigen(HBeAg)expressions in Huh7 cells. Methods The full length genotype B and C HBV DNA were extracted and amplified from two HBV infected patients. The recombinant plasmids were constructed by inserting the amplified HBV fragments into the eukaryotic expression vector,pHY106,which were then transfected into Huh7 cells. The cells transfected with blank pHY106 vector were used as control. HBV DNA replication at 72 hours of transfection was detected by Southern blot. The HBV DNA levels in Huh7 cells at 24,48,72,96 and 120 hours of transfection were determined by real-time polymerase chain reaction(PCR).Meanwhile, the HBsAg and HBeAg expression levels in the supernatants at 24,48,72,96 and 120 hours were determined by enzyme linked immunosorbent assay(ELISA).Results The recombinant plasmids expressing genotype B or C HBV DNA were successfully constructed.The HBV replicative intermediates in HBV core particles,including rcDNA dsDNA and ssDNA,were detected by Southern blot.HBV DNA level could reach 8 lg copy/mL which was by real-time PCR. HBsAg and HBeAg levels determined by ELISA peaked at 72 hours after transfection and then declined gradually. Conclusions The recombinant plasmids inserted with genotype B or C HBV DNA are constructed successfully, which can express high levels of HBsAg and HBeAg in Huh7 cells. This system provides a platform for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs against HBV.

Objective To investigate the effect of pathoglycemia on the tumor biomarker expression in pancreatic tumor and assess its influence on the diagnostic value of these biomarkers.Methods We recruited 59 patients with malignant pancreatic tumor in total into this study,including 46 cases with pancreatic carcinoma and 13 cases with other pancreatic malignancies.Twenty-seven patients with benign pancreatic diseases were selected as control.All subjects were extracted venous blood and serum samples were separated by centrifugation.Serum levels of CA199,CA125 and CEA were measured by chemiluminescent microparticle immunoassay(CMIA).The positive expression criteria were designated as ＞37 kU/L for CA199;＞35 kU/L for CA125;＞10 μg/L for CEA.Results (1) CA199 showed the highest diagnostic accuracy when the tumor biomarker was used alone.CA199 plus CEA showed the highest diagnostic accuracy when the biomarkers were used pairwisely,even better than three biomarkers used in combination(x2=26.131,P＜0.05).(2)The average rank of all tumor biomarkers were higher in all malignant pancreatic tumor than benign pancreatic diseases,and some of the differences reached statistically significance(CA199,Z=4.682,P=0.000;CA125,Z=1.866,P=0.062;CEA,Z=2.573,P=0.010).(3)When the malignant pancreatic tumor group were further divided into two groups according to their blood sugar level,we found that CA199 were significantly higher in pathoglycemia group than euglycemia group(Z=2.265,P=0.024),while no significant differences were observed in patients with benign pancreatic diseases when compared between patients with different blood sugar levels(Z=4.214,3.224,3.154,Ps＞0.05).Conclusion The combination use of three tumor biomarkers showed no improvements in the diagnosis of pancreatic neoplasm,but with disadvantage of elevated medical expense.CA199 showed the highest diagnostic accuracy when used alone.CA199 plus CEA showed the highest diagnostic accuracy when the biomarkers were used pairwisely.The blood sugar level should be considered when using CA199 in the differential diagnosis of pancreatic neoplasm from benign pancreatic lesions;a new set-point of CA199 should be set by studying the ROC curve and other statistic index to improve the overall accuracy.

Objective To construct the recombinant plasmid pEGFP-N1-SrV+ and evaluate the expression of SrV+in mammalian 293T cells.nethods srv+.a gene encoding the vailable region of the surface protein of the Streptococcus mutans OMZ175.was cloned chemically based on its reported nucleotide sequence.The eukaryotic expression plasmid,pEGFP-N1-SrV+,was constructed by introducing the srv+ gene into the Kpn Ⅰ/Xho Ⅰ site of pEGFP-NI.The recombinant plasmid pEGFP-N1-SrV+was transfected into 293T cells with lipofectamine and the expression level of SrV+was evaluated.Results The eukaryotic expression plasmid pEGFP-N1-SrV+was constructed successfully.GFP was observed by green fluorescent microscope.and a 72 × 1 03 protein was detected bv Westem blot.Real-time RT-PCR analysis revealed that the expression of the pEGFP-N1-SrV+in 293T was excellent and significant compared the control group. Conclusion The recombinant plasmid pEGFP-N1-SrV+was successfully constructed.which could encode the expression of SrV+after transfected into the mammalian 293T Cells.

Objective To understand and to grasp the basic situation on blood donors who were ineligible by ALT dry chemistry testing before blood donation.Methods The ALT rejection ratios used in initial screening and not used in two blood collection point were compared at the same time.The relativity between the dry chemistry testing and rate method were compared.An association was explored in various influencing factors on the activity of ALT.Results The use of ALT initial screening in blood donation had a significantly lower unqualified rate than not using it in early screening (P＜0.05).Dry chemical,biological method and the results had good correlation rates (r=0.993,P＜0.05).The B MI,drinking,sleeping,sports,diabetes,liver disease were independent factors affecting ALT activity (P＜0.05).Conclusion ALT initial screening can significantly reduce the unqualified rates and disposal of blood.

Objective To explore characteristics and treatment of the clinical crises myasthenia sufferer.Methods Retrospective analysis 32 patients with clinical data of MG crises,intravenous immunoglobulin all.Results Treating group,recovered in 1 case,the basic recovery in 4 cases,markedly effective in 6 cases,improved in 3 cases,Ineffective in 2 cases;the total effective 31.25%.Conclusion Group,recovered in 0 case,the basic recovery in 1 cases,markedly effective in 2 cases,improved in 3 cases,Ineffective in 10 cases; efficient 37.50%.Two groups of efficient comparative greup(X2 = 4.54 ,P <0.05) conclusion:Improved the patients's clinical symptoms after intravenous injection of gamma globulin,and the titer of patients with blood AchRAb significantly lower than that before treatment.

BACKGROUND:Studies have shown that there are large differences in the thickness of the soft tissue overlying hard tissue, and the soft tissue does not uniformly overly the hard tissue, indicating simple hard tissue measurement wil not harvest ideal facial profile in clinical treatment of malocclusions. OBJECTIVE: To study the craniofacial soft and hard tissue characteristics in the adult Angle class II malocclusion, and then to analyze the relationship between Angle class II1 and class II2 malocclusions. METHODS: Sixty patients with adult Angle II malocclusion who were accepted by the Department of Orthodontics of Stomatological Hospital Affiliated to Jiamusi University from 2011 to 2014, on gender parity, aged 18-38 years (mean age of 26.3 years), including 30 cases of Angle class II1 and 30 cases of Angle class II2. Differences between the adult Angle class II1 and class II2 malocclusion patients were compared by cephalometric analysis based on X-ray measurement. Statistical correlation analysis was performed.RESULTS AND CONCLUSION:(1) Comparisons of hard tissue measurement of adult Angle class II1 and Angle class II2 malocclusions showed that: SNB, SND, ANB, FH-NP, U1-SN (P < 0.001), LI-NB (P< 0.01), L1-MP (P < 0.01), U1-L1 (P < 0.001) exhibited statisticaly significant differences between two groups (P < 0.05). (2) Comparisons of soft tissue measurement of adult Angle class II1 and Angle class II2 malocclusions showed that: there were significant differences in the ULA'-FH, LLNs-FH, ULNs-FH, CmSnUL, E-LL (P < 0.05). (3) There was a correlation between the soft and hard tissue of adult Angle class II1 and Angle class II2 malocclusions in al measurement indexes, but the correlativity exists differently. These findings indicate that for Angle class II1malocclusion, the maxilary and anterior teeth protrusions have a certain influence on the position of the lower lip; for Angle class II2 malocclusion, only maxilary protrusion can impact the position of the soft tissue of the lower lip. Chin soft tissue has no major changes in Angle class II2 malocclusion, but it varies greatly in Angle class II1 malocclusion. Clinical treatment of adult Angle class II malocclusions is developed based on the craniofacial soft and hard tissue characteristics in orthodontic and orthognathic surgeries.

Objective To detect K-ras mutations in plasma by a nano capture probe system , and to explore the diagnostic and prognostic value of this method for patients with pancreatic cancer .Methods The clinical data of 62 patients with pancreatic cancer , 38 patients with benign pancreatic diseases and 31 healthy controls admitted in the Second Affiliated Hospital of Jiaxing Medical College from June 2013 to June 2015 were collected.The diagnosis of all the patients were confirmed by pathology .The DNA were extracted from all plasma samples and were detected for the codon 12 and 13 mutation of K-ras gene by nano capture probe and conventional PCR plus direct sequencing .The correlation of K-ras gene mutation with certain clinical data and the diagnostic and prognostic value in pancreatic cancer were analyzed .Results The K-ras mutation were detected by nano capture probe in 27 pancreatic cancer patients , and the mutation rate was 43.5%(27/62), including 25 cases with codon 12 mutation and 2 cases with codon 13 mutation .The K-ras mutation rate in patients with benign pancreatic diseases was 7.9%(3/38), which were all in codon 12.K-ras mutation was detected in only 17 pancreatic cancer patients by conventional PCR plus direct sequencing , and the mutation rate was 27.4%(17/62), The K-ras mutation rate of benign pancreatic diseases was 5.3%(2/38).The mutation rate detected by nano capture probe was higher than that by conventional PCR , and the difference was statistically significant (P=0.006).K-ras mutation in the plasma of patients with pancreatic cancer was related to TMN stage and liver metastasis , but there was no correlation of the factors such as sex , age, clinical symptoms, tumor size, serum CA19-9 and CEA levels with K-ras mutation.The sensitivity of K-ras gene mutation for diagnosing pancreatic cancer was 43.5%, the specificity was 92.1%, the positive predictive value was 90%, the negative predictive value was 50%, Youden index was 0.356.The 1-year survival rate of patients with K-ras mutation was 44.4%, which was lower than that (71.4%) of patients with wild-type K-ras, and the difference was statistically significant (P<0.05).Conclusions The nano capture probe system could certainly detect K-ras mutation in a small quantity of plasma DNA , and its diagnosis sensitivity for pancreatic cancer is low , but the specificity is relatively high .K-ras mutation in plasma is closely related to the TMN stage and prognosis of pancreatic cancer .

Background and purpose: Pancreatic intraepithelial neoplasia (PanIN) may be a precursor lesion of inifltrating pancreatic ductal adenocarcinoma. The mutation of the phenotypic impact of K-ras G12D alone, silencing of p53 and p16 could promote this process. The role of Smad4 in this progression was poorly understood. In our previous studies, we investigated that RNA interference silence of Smad4 to promote the PanIN cell malignant transformation. In the present study, we investigate. The further explores the siRNA interference of Smad4 expression on PanIN cells could lead to proliferation and metastasis in vitro and in vivo. Methods:Smad4 knock-down PanIN cells (PanIN-S) were established by stable transfection with lentiviral-mediated Smad4 RNA interference. In vitro,silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. A soft agar assay was used to assess the anchorage-independent growth ability of cells. Cell migration and invasion assays were performed using transwell chambers with or without Matrigel. In xenograft model experiments, PCNA, VEGF and MMP-9 staining was separately used to evaluate cell proliferation and angiogenesis and migration (VEGF and MMP-9). Results:Effect of siRNA of Smad4 gene in PanIN cells was conifrmed by real-time RT-PCR and western blot. In vitro, silence of Smad4 enhanced the proliferation of PanIN cells as determined by cell counting. Soft agar assay showed that there were more colony cell numbers in PanIN-S cells compared with PanIN cells (P<0.05). Using the transwell assay, we observed that PanIN-S cells migrated faster than PanIN cells and similar results were obtained by Matrigel assay (P<0.05). Furthermore, immunohistochemical analysis of the harvested tumors suggested that Smad4 silencing was associated with cell proliferation (PCNA reactivity) and angiogenesis and migration (VEGF and MMP-9), and the expressions of PCNA, VEGF and MMP-9 in PanIN-S group were signiifcantly increased (P<0.05). Conclusion:Silence of Smad4 in PanIN cells enhanced progression to invasive adenocarcinoma of the pancreas by promoting cell growth, migration and invasion. Smad4 might be a new diagnostic marker in pancreatic cancer and prove to be a feasible and novel target for therapeutic intervention.

Objective To express nonstructural protein 2 transactivated protein (NS2TP) of hepatitis C virus (HCV) in the prokaryotic expression system and prepare polyclonal antibody,and to study the expressions in different liver tissues.Methods NS2TP gene was amplified by polymerase chain reaction (PCR) technique and cloned into the prokaryotic expression vector pET-32a(+),which was transformed into E.coli BL21.The protein was induced by isopropyl thiogalactose (IPTG) and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blotting assay.The recombinant protein were expressed and purified in a large amount.The rabbit was immunized with the purified protein to prepare polyelonal antibody.The liver tissues of patients with chronic HCV infection and healthy controls were detected by immunohistochemistry method.Results The recombinant NS2TP protein (relative molecular mass:33×103 ) and polyclonal antibody with high titer and specificity were successfully prepared.NS2TP expressions in the liver of patients with chronic HCV infection were higher than those of healthy controls,and were mainly distributed in the nucleus of hepatocytes.Conclusions The NS2TP expression level and intracellular location in liver tissue of patients with chronic HCV infection are understanded,which could bring new clues for further study of the biological function of NS2TP and the pathogenesis of HCV infection.

Objective To explore the value of quantitative T2 weighted magnetic resonance imaging (T2WI) and diffusion tensor imaging (DTI) parameters for predicting functional outcomes after surgery for cervical spondylotic myelopathy (CSM).Methods One hundred and forty CSM patients received T2WI and DTI before surgery at Tianjin Hospital between April 2014 and April 2016.They were then given systematic rehabilitation treatment after the surgery.The Japanese Orthopaedics Association (JOA) scoring system was applied to evaluate their neurological function before and after the surgery.According to the JOA recovery rate at 1-year follow-up,the patients were divided into a good recovery group (recovery rate≥ 50％) and a poor recovery group (recovery rate ＜50％).Maximum spinal cord compression (MSCC),signal change ratio (SCR),transverse area (TA),apparent diffusion coefficient (ADC) and fractional anisotropy (FA) were compared between the two groups.Receiver operating characteristics (ROC) curves were used to measure the prognostic ability and determine the best cut-off value for each variable.The independent predictors of a poor recovery were estimated using univariate and multivariate analysis.Results ROC analysis showed that the area under the ROC curve (AUC) of MSCC was 0.593.For SCR it was 0.682.For the TA it was 0.706.For the ADC it was 0.719 and for FA it was 0.749.The respective cut-off values were 44％ compression,1.561,0.46 cm2,1.339×10-3 mm2/s and 0.386.FA had the largest AUC,followed by ADC,TA,SCR and MSCC.MSCC and SCR had low discrimination power (AUC＜0.7) in predicting a poor recovery,whereas TA,ADC and FA had moderate discrimination power (AUC 0.7-0.9).Logistic multivariate regression showed that a low JOA score,TA≤0.46 cm2 or FA≤0.386 were independent risk factors for a poor recovery.A predicting model built according to the results of the logistic regression analysis gave an AUC of 0.87,significantly better than that of the JOA score.With a cut-off value of 0.36,the sensitivity and specificity were 80％ and 77％ respectively.Conclusions Combining T2WI and DTI parameters with the JOA score may better predict the recovery of patients with CSM.The values can also provide references for making up rehabilitation plans.