Objective To develop a quantitative immunohistochemistry assay for duck hepatitis B virus core antigen (DHB-cAg)in duck liver tissue.Methods By comparison with no repair antigen and repair antigen with high pressure,microwave and trypsin,the best solution of antigen retrieval was determined.By optimizing the parameter of image acquisition and de-ducting blank area,mean density of yellow areas was calculated using Image-Pro Plus 6.0 software.Using the assay devel-oped to determine the level of DHBcAg in liver tissue from duck infected by DHBV,anti-DHBV activity of DHBcMAb-TAT PTD conj ugate was examined.Results SABC method with no repair antigen was selected,which was better than other methods.DHBcAg expression in duck liver tissue could be objectively and accurately quantified by setting Image-Pro Plus 6.0 software parameters and calculating mean density of yellow areas.By comparison with the differences between mean densityat baseline of treatment and end of treatment,it was showed that DHBcMAb-TATPTD conjugate treatment dose-de-pendently reduced the levels of DHBcAg in liver tissue,which show that the assay developed could effectively evaluate the anti-DHBV activity of agent.Conclusion The immunohistochemistry assay developed in this study can objectively and accu-rately evaluate the level of DHBcAg in duck liver tissue.

Objective To explore the expression of T cell immunoglobulin and mucin-3(TIM-3)in peripheral blood mononu-clear cells from patients with primary Sj¨ogren’s syndrome(pSS)and its clinical significance.Methods Enrolled 33 pSS pa-tients from Shanghai Changzheng Hospital and Changhai Hospital in October 2012 and July 2013.The relative expression of TIM-3 in the peripheral blood mononuclear cells(PBMC)in 33 patients and 33 healthy individuals was detected by RT-PCR. The association between TIM-3 mRNA level and clinical characteristics were analyzed,for example,serum RF,CRP and anti-SSA/SSB antibody.1 2 of the pSS patients were followed up and their TIM-3 mRNA level was determined after glucocorti-coid treatment for 2 months.Results The expression of TIM-3 in PBMCs from patients with pSS was increased significantly than that in healthy individuals (0.55±0.12 vs 0.13±0.10,t=15.44,P<0.000 1),but its expression between anti-SSA/anti-SSB positive and negative patients were not statistical difference (0.45±0.21 vs 0.54±0.31;0.46±0.15 vs 0.51± 0.24).The TIM-3 mRNA expression in pSS patients was positively correlated with serum RF and CRP (R=0.558 5,R=0.414 7,P>0.05),but was not associated the anti-SSA/SSB antibody (P=0.298 2).TIM-3 mRNA level was decreased in the patients who were treated with glucocorticoids for 2 months (0.62±0.10 vs 0.38±0.13,t=5.07,P<0.05).Conclusion TIM-3 may play a role in the pathogenesis of pSS and it may be regarded as a biomarker to pSS.

Objective To investigate the mutation screening of the GJB3,GJB2,mtDNA 1555 A>G and SLC26A4 gene in Hainan Pronive population with non-syndromic hearing impairment.Methods PCR were performed with one pair of primer in the coding sequence of GJB3,GJB2,mtDNA 1555 A>G and SLC26A4 gene.Bidirectional sequencing of PCR products was subsequently applied in 429 patients with hearing loss.Results 55 patients gene mutation of 429 patients were found. The point mutation in mtDNA was found in 5 patients (1.1 7%).1 5 5 5 A>G mutation of mtDNA was found in 4 patients. 1494 C>T mutation of mtDNA was found in one patients.GJB2 gene mutation was found in 25 patients (5.83%).235 del C mutation of GJB2 gene was found in 9 patients.235 del C/GJB2 299 del AT mutation was found in two patients.235 del C mutation was found in 10 patients.176 del 16 mutation was found in 4 patients.SLC26A4 mutation was found in 22 patients (5.13%).IVS7-2 A>G mutation of SLC26A4 was found in 4 patients.2168 A>G mutation of SLC26A4 was found in one patient.IVS7-2 A>G mutation was found in 12 patients.2168 A>G mutation was found in 5 patients.538 C>T mutation of GJB3 gene was found in 3 patient.IVS7-2A>G mutation and 2168 A>G muation of SLC26A4 gene was found in 4 of 22 EVA patients.Conclusion GJB2 gene and SLC26A4 gene have revealed responsible genes for Hainan deafness patients.

Objective To verify the reference interval for D-dimer assay and analyze the influence of age and gender on the ref-erence interval.Methods Inclusion criteria for reference individuals were established.60 healthy males and 63 females were enrolled and divided to three groups by age,including 20 to 39 years old group (20 males and 20 females),40 to 59 years old group (20 males and 23 females)and above 60 years old group (20 males and 20 females).Blood samples were drawn in cit-rate sodium anticoagulated tubes and D-dimer concentration was determined by three different coagulation analyzers using o-riginal reagents.According to CLSI guideline C-28-A3,the reference interval for each measurement system from reagent manufacturer was verified and the difference of D-dimer concentration between different age-group and sex-group was ana-lyzed using non-parameters tests.Results All reference intervals were verified for people under age 40,while one reference interval cannot be verified for people from 40 to 59 years old as same as one for people above 60 years old.D-dimer concen-tration increased with age and there was significantly different between 20~39 years old group and 40~59 years old group or above 60 years old group(P<0.05).There was only a significant difference between sex-group for people under age 60(P<0.05).Conclusion D-dimer concentration was associated with age and sex.For people under age 40,the reference inter-val from reagent manufacture can be verified and directly used in laboratory,while for people above age 60,the reference in-terval from reagent manufacture cannot be verified.The cause should be investigated and a new reference interval should be established separately when necessary.

Objective To explore TRAIL expression in patients with primary immune thrombocytopenia (ITP)and its role in the pathogenesis.Methods Peripheral Blood Mononuclear Cells (PBMCs)from twenty-eight patients with ITP and thirty healthy controls were obtained.TRAIL expression was determined using Real-time Quantitative Polymerase Chain Reaction (RT-PCR).Serum TNF-α,IFN-γ,IL-4 and IL-10 were detected using ELISA.Associations between TRAIL expression and clinical parameters were assessed.Results Compared to healthy controls,TRAIL expression was significantly increased in PBMC from patients with ITP (2.80±0.43 vs 1.00±0.24,t=19.72,P<0.001).Compared to the healthy controls,ex-pression of IFN-γand TNF-αfrom ITP patients were increased (52.43±11.04 pg/ml vs 23.26±8.15 pg/ml;69.14± 10.89 pg/ml vs 36.14±13.17 pg/ml,t=10.36~11.50,P<0.001)and expression of IL-10 were decreased (11.18±6.13 pg/ml vs 33.28±9.85 pg/ml,t=10.17,P<0.001)and there was no significant difference in IL-4 expression (35.75± 12.52 pg/ml vs 40.16±14.26 pg/ml,t=1.25,P>0.05).Furthermore,TRAIL expression was positively correlated with serum IFN-γ,TNF-αlevel (r=0.432,0.541,P<0.05),and negatively correlated with IL-10 and PLT (r=-0.424,-0.553,P<0.05).Conclusion Rur results suggest that TRAIL may be involved in the pathogenesis of ITP.

Objective To explore the association between the level of lactate dehydrogenase (LDH)in gastric cancer patients’ serum or the relative expression of LDH in their tissues and the patients’prognosis.Methods 60 specimens of gastric canc-er patients who confirmed by pathological diagnosis were collected from 2012 to 2013,include the serum specimens and tis-sue specimens.Their serum level of LDH were detected,and the relative expressions level of LDH in the sample of normal tissues,gastric cancer tissues and metastatic lymph nodes were quantify by real-time fluorescence quantitative reverse tran-scription-PCR (Realtime-PCR).Finally,the relationship between the expression of LDH and clinical pathologic features were analyzed by independent t-test or anova statistics.Results The serum level of LDH was highly increased than control group (340.89±10.67 IU/ml,t=24.7,P<0.01);the relative gene expression of LDH in normal tissue,human gastric cancer and metastatic lymph node were 1.0,3.39 and 2.35.The result suggests the serum level of LDH were associated with pTNM stage and lymph node metastases (t=5.2,4.8,P<0.01).The relative gene expression of LDH in gastric canc-er tissues were associated with tumor sizes,pTNM stage and lymph node metastases (t=18.2,15.3,P<0.01 and F=7.2, P<0.01).Conclusion The serum LDH level and the expression of LDH in cancer tissue were significantly increased,and their expression were correlated with the patient’s prognosis.The serum level of LDH and the expression of LDH in gastric cancer tissue may be potential indicator to evaluated the prognosis of the patient with gastric cancer.

Objective To develop TaqMan real-time PCR assay for detection and identification of streptococcus pneumonia iso-lated from children CAP.Methods Based on the sequences of lytA gene,primers and probe were designed and the assay was optimized.Then 1 504 sputum samples were detected by culture and the developed assay with double-blind testing.Results The lower limit of detection in the developed real-time PCR assay was 18.75 cfu/PCR,and had no cross reaction.141 strep-tococcus pneumonia strains were detected from 1 504 samples and 140 were isolated by culture.The whole process just nee-ded 2.5 h.Conclusion The established assay is rapid,simple,high sensitivity and specificity.It is not only valuable for the i-dentification of streptococcus pneumonia,but also provide evidence for antibiotic therapy.

Objective To explore the role of microRNA-206 (miR-206)in peripheral blood mononuclear cells (PBMC)from rheumatoid arthritis (RA)patients.Methods 27 patients with RA and 25 healthy controls were enrolled into the current study.Human peripheral blood mononuclear cells (PBMCs)were isolated by standard density-gradient centrifugation over Ficoll-Hypaque solution in rheumatoid arthritis and healthy control volunteers.Total RNAs were extracted from PBMCs which were stimulated by PMA and ionomycin,then the RNA was transcribed reversely into cDNA.The expression of mi-croRNA-206 and Kruppel-like factor 4 (KLF4)and the orphan nuclear receptor RORγt mRNA was detected by real-time quantitative PCR (qRT-PCR)method.Student’s unpaired t-test and spearman correlation were used for statistica1 analysis. Results The expression of miR-206 in the PBMCs of RA patients was significantly decreased compared with that of the healthy controls (0.056±0.019 vs 0.138±0.057,t=3.103,P<0.01),The levels of KLF4 and RORγt mRNAin the PB-MCs of RA patients were increased significantly verves those of the healthy controls(0.604±0.183 vs 0.098±0.027,t=6.651,P<0.01;0.583±0.271 vs 0.069±0.018,t=7.438,P<0.01),Furthermore,a negative correlated between the ex-pression of miR-206 and KLF4 or RORγt mRNA in RA patients (r=-0.639,P<0.01;r=-0.842,P<0.01).Conclusion These results indicated that the augmented expression of KLF4 mRNA may be caused by the attenuated expression of miR-206,and the high level of KLF4 mRNA evokes the proportion of Th17 cells in RA patients.

Objective To investigate the expression level of peripheral blood mononuclear cell(PBMC)human leucocyte anti-gen G(HLA-G)in patients with hepatocellular carcinoma(HCC).Methods The HLA-G mRNA in PBMC from 44 patients with HCC,21 patients with liver cirrhosis and 40 healthy subjects were measured by reverse transcription real time fluores-cent relative quantitative PCR.Results HLA-G mRNA expression level were 1.71±0.39,1.05±0.38 and 1.01±0.47 in HCC group,liver cirrhosis group and healthy control group respectively.HCC group was higher than the other two groups, the difference was statistically significant (F=33.657,P<0.001).The survival rate of HCC patients in HLA-G mRNA high-expression group was lower than HLA-G mRNA low-expression group,the difference was statistically significant (χ2=5.972,P=0.015).Conclusion PBMC HLA-G mRNA in HCC was closely correlated with tumorigenesis.It can proviede a novel diagnosis and research tool for HCC.

Objective To establish and apply the multiple probe RT-PCR analysis for H5N1 pathogen detection methods. Methods Used the gene sequences published in GenBank,which were designed and synthesized two pairs of specific primers (P1/P2,P3/P4)accorded the conserved regions.Through the optimization of amplification conditions to establish the rapid detection of two viruses duplex PCR method.Results The suitable primer concentration:P1/P2 was 0.32μmol/L,P3/P4 was 0.96μmol/L.The amount of the minimum virus nucleic acid was 2 ng/μl by the double detect,H5N1 pathogen mixed were occurred specific bands in the corresponding location,and other viruses and blank control strips had not bands.Conclu-sion A variety of probes for RT-PCR analysis H5N1 pathogen detection methods in a reasonable application of primer con-centration under has good sensitivity and specificity,and it is worth the inspection application.