Based on the gene sequence of AIV matrix protein 2(M2) and two cytotoxic T-lymphocyte epitopes derived from nucleoprotein,the prokaryotic expression vector pET-3M2e-NP1-NP2 was constructed.The target gene was expressed in the solvable form in BL21(DE3) when induced with 1.0 mmol/L IPTG and Western blot analysis showed that the expression product had good immunogencity.Purified fusion protein was mixed with various amount of adjuvant,including Freund's,Vash oil and chitosan,and then immunized 20-day-old chicken by intramuscular injection and boosted 3weeks later.Blood samples were collected weekly following the primary vaccination.The anti-M2e antibody was detected with ELISA method with the synthesized peptide as coated antigen;the neutralizing ability of anti-serum was evaluated on MDCK cell line and chick embryo,the CD4+ and CD8+ T lymphocyte amounts in peripheral blood of immunized chicken was measured by flow cytometry.Results showed that the fusion protein can induce immunological reaction,and the antibody can bind with the viral M2 protein that expressed on the surface of MDCK cells.Flow cytometry result showed that CD4+ and CD8+ T lymphocyte in peripheral blood increased obviously following immunization(P

M2e gene of three copies for H5N1 subtype AIV was synthesized and fused with human mycobacterium tuberculosis hsp70 gene.The fused gene was cloned into the prokaryotic expression vector to get pET-3M2e and pET-3M2e-hsp70.Recombinant protein r3M2e and r3M2e-hsp70 were successfully expressed induced with IPTG and purified with Ni2+-NTA collumn.Following that,the immunity of the recombinant protein was analysized with Western blot.20-day-old AIV non-immunized chickens were vaccined with r3M2e and r3M2e hsp70,at the same time,Trx and KLH-M2e inoculated chickens were served as vector and positive controls.Two weeks after the primary vaccination,every group was boosted with the same vaccine as in the primary vaccination.The humoral immunity of the vaccined chickens was evaluated with antibody detection against M2e,cytopathic suppression test,and indirect fluorescence assay.The cellular immunity was estimated according to lymphocyte subtype analysis with flow cytometry and M2e specific cytokine detection.Four weeks after the boost vaccine,all groups were challenged with 100EID50 AIV of H9N2 subtype,and the virus from swabs was detected with Real-time PCR.Results indicated that r3M2e hsp70 vaccined chicken developed the better humoral and cellular immune response,also,made a better performance compared with r3M2e vaccined group in virus challenge.

OBJECTIVETo evaluate the feasibility of multi-slice spiral CT scan to localize upper airway stricture in patients with obstructive sleep apnea syndrome (OSAS) during drug-induced sleeping.

METHODSOne hundred and fourteen patients diagnosed as OSAS by polysomnography were included in the study. Multi-slice spiral CT scan covering upper airway was performed at the end of inspiration and clear upper airway images were obtained in waking. After injecting 5 mg of midazolam intravenously slowly in 109 patients, CT scan was performed at apnea and clear upper airway images were obtained in sleeping. Cross-section area and minimal diameter of airway were measured and the parameters were compared under those two states. Upper airway was displayed intuitionisticly by using post-processing techniques.

RESULTSOne hundred and nine patients with OSAS finished the examination with a success rate of 100 %. Airway obstruction at retropalatal level was observed in 62 patients, among whom 26 were associated with airway obstruction at retroglossal level, 27 with narrower airway at retroglossal level in sleeping compared with that in waking, and 9 with no significant change of the airway at retroglossal level after sleeping. Narrower airway at retropalatal level in sleeping compared with that in waking was observed in 40 patients, among whom 20 were associated with narrower airway at retroglossal level in sleeping compared with that in waking, 10 with complete airway obstruction at retroglossal level in sleeping, and 7 with no significant change of the airway at both retropalatal and retroglossal levels before and after sleeping. Minimal mean cross-section area of airway at retropalatal level was (72.60 +/-45.15)mm(2) in waking and (8.26 +/-18.16)mm(2) in sleeping; and minimal mean cross-section area of airway at retroglossal level was (133.21 +/-120.36)mm(2)in waking and (16.73 +/-30.21)mm(2) in sleeping (P <0.01). Minimal mean diameter of airway at retropalatal level was (6.91 +/-2.23) mm in waking and (1.18 +/-2.14) mm in sleeping; and minimal mean diameter of airway at retroglossal level was (8.68 +/-4.32) mm in waking and (1.68 +/-2.22) mm in sleeping (P <0.01).

CONCLUSIONMulti-slice spiral CT with post-processing techniques can display the shape of the upper airway in patients with OSAS in sleeping, and can localize the upper airway stricture and assess its range accurately.

Adult ; Aged ; Airway Obstruction ; diagnostic imaging ; Female ; Humans ; Hypnotics and Sedatives ; administration & dosage ; Male ; Middle Aged ; Oropharynx ; physiopathology ; Palate, Soft ; physiopathology ; Sleep Apnea, Obstructive ; diagnostic imaging ; Tomography, Spiral Computed ; Young Adult

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo establish chemiluminescence enzyme immunoassay (CLEIA) for quantitative detection of procollagen III N-terminal peptide (P III NP) in serum.

METHODSA sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of P III NP as the catalytic enzyme and the luminol as the luminescence reagent. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on. The CLEIA was compared with imported ELISA kits, by detecting clinical serum.

RESULTSThe linear range was 0.8-85 ng/ml. The detection limit was 0.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 96.2%, 91.2% and 101.1%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.99 and RSD lower than 6%. The detected results of clinical sera with CLEIA closely corresponded to those with imported ELISA.

CONCLUSIONEstablished CLEIA for quantity determination of serum P III NP has high accuracy, sensitivity and repeatability.

Adult ; Aged ; Antibodies, Monoclonal ; analysis ; Enzyme-Linked Immunosorbent Assay ; instrumentation ; methods ; Female ; Humans ; Liver Cirrhosis ; blood ; diagnosis ; Luminescence ; Luminescent Measurements ; methods ; Male ; Middle Aged ; Peptide Fragments ; blood ; chemistry ; Procollagen ; blood ; chemistry ; Sensitivity and Specificity

OBJECTIVETo establish enzyme-linked immunosorbent assay (ELISA) for quantitative detection of Golgi protein73 (GP73) in serum.

METHODSA sandwich reaction was preformed with horseradish peroxidase labeled monoclonal antibody of GP73 as the catalytic enzyme. Several reactions liquid's concentration and reaction conditions were optimized. The method was evaluated in all aspects such as linear range, sensitivity, specificity, stability and so on.

RESULTSThe linear range was 25-500 ng/ml. The detection limit was 18.5 ng/ml. Inter-assay and intra-assay RSD were both less than 10%. The recoveries of three different spiked concentration samples were 95.3%, 92.6% and 103.7%. After stored at 4 degrees C and 37 degrees C for 3, 5, 7 days, the analysis showed correlation coefficient higher than 0.98 and RSD lower than 10%.

CONCLUSIONEstablished ELISA for quantity determination of serum GP73 has high accuracy, sensitivity and repeatability.

Antibodies, Monoclonal ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Membrane Proteins ; blood ; Sensitivity and Specificity

OBJECTIVETo investigate the association of SOX9 expression and clinicopathologic factors and prognosis of gastric cancer.

METHODSA retrospective cohort study including 112 gastric cancer patients admitted to the Zhejiang Provincial People's Hospital from 2004 to 2006 was performed. Immunohistochemical analysis was used to evaluate the expression of SOX9 in the 112 specimens of gastric cancer tissues and 70 non-cancerous tissues adjacent to the tumor.

RESULTSLow expression of SOX9 was seen in 5(7.1%) tissues out of 70 non-cancerous tissues adjacent to the tumor. A total of 94(83.9%) patients had varying expression of SOX9, of whom 51(45.4%) had overexpression. Univariate analysis demonstrated that the expression of SOX9 was significantly associated with Lauren classification (P<0.05), tumor invasion(P<0.01), lymph node metastasis(P<0.05), distant metastasis(P<0.05) and tumor stage(P<0.05), however there was no significant association between SOX9 expression and sex, age, histological type, histology differentiation or tumor size. Kaplan-Meier analysis showed that the 5-year survival rate of patients with SOX9 over-expression was significantly lower than that of patients with low expression(29.4% vs. 49.2%, P=0.031). Multivariate Cox regression analysis showed that histology differentiation(P=0.046), tumor invasion(P=0.001), and distant metastasis(P<0.01) were independent prognostic factors for gastric cancer, however the over-expression of SOX9 was not significant(P=0.948).

CONCLUSIONSThe expression SOX9 is associated with the growth, invasion, and metastasis of gastric cancer, as well as the prognosis. However, SOX9 expression is not an independent factor for the prognosis in patients with gastric cancer.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; SOX9 Transcription Factor ; metabolism ; Stomach Neoplasms ; metabolism ; pathology

OBJECTIVETo screen genes differentially expressed in mouse hepatocarcinoma ascites cell line with high potential of lymphatic metastasis.

METHODSA subtracted cDNA library of mouse hepatocarcinoma cell line with high potential of lymphatic metastasis Hca-F and its synogenetic cell line Hca-P with low metastatic potential was constructed by suppression subtractive hybridization (SSH) method. The screened clones of the subtracted library were sequenced and GenBank homology search was performed.

RESULTSTen differentially expressed cDNA fragments of Hca-F with high potential of lymphatic spreading were obtained, two of which were newly identified ones.

CONCLUSIONSSH is a useful technique to detect genes of differential expression and an effective method to clone novel genes.

Animals ; Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Gene Library ; Liver Neoplasms, Experimental ; genetics ; pathology ; Lymphatic Metastasis ; genetics ; Mice ; Mice, Inbred Strains ; Nucleic Acid Hybridization ; methods ; RNA, Messenger ; genetics

Objective: To investigate the joint effect of abdominal obesity in childhood and adulthood on adult hypertension. Methods: Based on the data from the China Health and Nutrition Survey (CHNS, 1993-2011), a total of 1 431 subjects who were investigated in both childhood (6 to17 years old) and young adulthood (18 to 35 years old) were selected for the analysis. According to waist circumference (WC) status (normal WC or abdominal obesity) in childhood and adulthood, all subjects were categorized into 4 groups. The multinomial logistic regression model was used to analyze the joint effect of abdominal obesity in childhood and adulthood on adult hypertension and pre-hypertension. Results: Compared to the subjects (n=1 057) who had normal WC in both childhood and adulthood, subjects with abdominal obesity in childhood but with normal WC (n=45) in adulthood didn′t have significantly increased risk of hypertension (OR=1.52, 95%CI: 0.19-12.06). In contrast, those who had normal WC in childhood and abdominal obesity in adulthood (n=289) had increased risk of hypertension (OR=6.48, 95%CI: 3.60-11.66). In addition, subjects with persistent abdominal obesity from childhood to adulthood (n=40) had the highest risk of hypertension (OR=15.98, 95%CI: 5.39-47.35). There was a similar trend for the association of abdominal obesity in childhood and adulthood with adult pre-hypertension, with the corresponding OR (95%CI) of 1.28 (0.66-2.49), 2.90 (2.17-3.89) and 3.49 (1.65-7.40), respectively. Conclusion The joint effect of abdominal obesity in childhood and adulthood increased the risk of adult hypertension and pre-hypertension. There was no statistical significance for subjects who had abdominal obesity in childhood but had normal WC in adulthood when compared to those with normal WC in both childhood and adulthood.

Objective To investigate the safety and effectiveness of open radical prostatectomy (ORP) for locally advanced prostate cancer (LAPC).Methods From January 2012 to April 2017,132 cases underwent ORP were included.The mean age was 65.1 years old (ranged 41 to 83 years old),median PSA was 28.9 ng/ml (ranged 1.2 to 319.7 ng/ml) and mean Glcason score was 8.0(ranged 6.0 to 10.0).The number of clinical stage T3aN0,T3bN0,T4N0 and T1 ～4N1 were 92 cases(69.7％),20 cases (15.2％),8 cases (6.1％) and 12 cases (9.0％),respectively.Results The median length of hospital day,mean operative time and median blood loss were 9 d,180 min and 350 ml respectively.The intraoperative complication rate was 3.0％ (4/132),including 2 rectum injury and 2 iliac vessel injury.Pathological tumor stage revealed that ≤ pT2 N0 7 cases (5.3％),pT3a N0 61 cases (46.2％),pT3b N0 38 cases (28.8％),pT4N0 12 cases (9.1％) and pT1～4N1 14 cases (10.6％).The mean Gleason score was 8.0 (ranged 6 tol0).The numbers of patients with perineural invasion,seminal vesicle invasion and positive surgical margin were 81 cases (61.4％),49 cases (37.1％) and 41 cases (31.1％) respectively.The median follow-up duration was 24.1 (ranged 1.8 to 62.2) months.The rate of postoperative complications was 3.0％ (4/132) including 1 urethral stricture,1 wound infection,1 intestinal fistula and 1 lymphatic fistula.The rates of patients with urinary continence 1,3,6 and 12 months after surgery were 30.4％ (38/125)、63.9％ (76/119)、72.6％ (82/112)、89.1％ (90/101).The rates of adjuvant hormonal therapy and radiotherapy were 34.1％ (45/132) and 38.6％ (51/132).One patient (0.8％) died of lung cancer.The rate of biochemical recurrence(BCR) was 25.8％ (34/132).The 5-year BCRfree survival rate was 57.2％ (95％ CI 41.9％ ～ 70.6％).Conclusion The oncological control and functional recovery outcomes of ORP for locally advanced prostate cancer were reliable.