Objective To investigate the value of tumor M2 pyruvate kinase (TuM2-PK ) ,cytokeratin-18 (CK18 )-3A9 and cytokeratin 19 fragment(CYFRA21-1) in non small cell lung cancer(NSCLC) ,and to evaluate the application value of combined de-tection of these three kinds tumor markers in diagnosis of NSCLC .Methods The serum levels of TuM2-PK and CK18-3A9 were measured in 67 patients with NSCLC(NSCLC group) ,72 patients with benign lung diseases(benign group) and 75 healthy control (control group) by enzyme linked immunosorbent assay(ELISA) .The level of CYFRA21-1 was detected by electrochemilumines-cence method .Results The serum levels of TuM2-PK ,CK18-3A9 and CYFRA21-1 in NSCLC group were higher than those in be-nign group and control group(P<0 .05) .The sensitivity of TuM2-PK or CK18-3A9 was better than CYFRA21-1(P<0 .05) ,the specificity of CYFRA21-1 was better than TuM2-PK or CK18-3A9(P<0 .01) .The sensitivity and accuracy of combined detection of TuM2-PK ,CK18-3A9 and CYFRA21-1 were both better than CYFRA21-1(P<0 .05) .The sensitivity ,specificity and accuracy of combined detection of TuM2-PK ,CK18-3A9 and CYFRA21-1 were all better than TuM2-PK or CK18-3A9 alone(P<0 .05) .Con-clusion The combined detection of TuM2-PK ,CK18-3A9 and CYFRA21-1 were better in sensitivity and accuracy of diagnosis of NSCLC .

Objective:To construct a recombinant retroviral vector carrying human inducible costimulator (ICOS) gene,screen for CHO cell line stably expressing ICOS protein and to study its biological activity.Methods: ICOS cDNA was obtained from human peripheral blood mononuclear cells (PBMC) through RT-PCR and was cloned into retroviral vector to construct retroviral recombinant pMSCV-ICOS; the latter was then packed and the high-titer virus producing cells were screened.Then CHO cell was infected by this high-titer virus and the stable cell line was screened.CHO-ICOS cells were co-cultured with PBMC (the ratio of CHO-ICOS to PBMC being 11,12,15, and 110) in presence of substimulating dose of anti-human CD3 antibody.The proliferation of PBMC and the CD25 expression on T cells were examined by 3H-TdR incorporation method and flow cytometry,respectively.CHO-pMSCV cells co-cultured with PBMC (11) served as the negative control and PBMC served as blank control.Results: We successfully constructed the retroviral recombinant pMSCV-ICOS and obtained CHO cell line stably expressing ICOS protein.3H-TdR incorporation method and flow cytometry showed that,compared with the negative control group and the blank control group,co-culture with CHO-ICOS cells significantly inhibited the anti-CD3 antibody-induced activation and proliferation of PBMC(P

Objective To investigate the serum levels of PGⅠ,PGⅡ,TK1,TSGF and CEA,CA724 in gastric cancer and eval-uate the application value of combined detection the above tumor markers in diagnosis of gastric cancer.Methods The serum levels of TSGF were measured in 94 patients with gastric cancer and 85 healthy control by rate method.PGⅠ,PGⅡ,TK1 and CEA,CA724 were detected by electrochemiluminescence method.Results PGⅠand PGⅠ/PGⅡwere lower than healthy control in serum of patients with gastric cancer (both P<0.05).There was no difference in PGⅡ (P>0.05),and other tumor markers were all higher than healthy control (all P<0.05).The sensitivity of PGⅠ,PGⅠ/PGⅡ were better than TK1,CEA and CA724 (all P<0.05),the specificity of PGⅠ/PGⅡ,CEA were better than TSGF (both P<0.05),the accuracy of PGⅠ,PGⅠ/PGⅡ were better than CA724 and TSGF alone (all P<0.05).When combined TSGF,TK1 and PGⅠ/PGⅡ,PGⅠ,the sensitivity was better than combined PGⅠ/PGⅡand PGⅠ alone (P<0.05).Then when added CEA, CA724,this sensitivity improved up to 82.98%.Although the combined detection would show a lower specificity,it still keep high to 84.71%.Combined detection improved the accuracy in diagnosis of gastric cancer,up to 83.80%.In this re-search,There was no difference in sensitivity,specificity and accuracy between the group of PGⅠ+PGⅠ/PGⅡ+TSGF+TK1+CEA and the group of PGⅠ+PGⅠ/PGⅡ+TSGF+TK1+CEA+CA724.Conclusion Compared with CEA and CA724 popular used in clinic,PGⅠ/PGⅡand PGⅠshowed a better application value.The group of PGⅠ+PGⅠ/PGⅡ+TSGF+TK1+CEA showed the best sensitivity.Combined detection of serum levels of PGⅠ,PGⅠ/PGⅡ,TSGF,TK1 ,CEA can significantly raise the sensitivity and accuracy in diagnosis of gastric cancer.

Objective To investigate the effect of vitamin D (VD) on macrophage to phagocytize Staphylococcus aureus (SA). Method Macrophoge cell lines RAW264.7 were allocated into 3 groups:control group(C),bacterium group(B),active vitamin D+ bacterium group (VD+B). Cells in the VD+B group were incubated with 10-8mol/L active vitamin D for 24h,then cells in the VD+B group and the B group were cultured with SA for 1h,and phagocytosis rate,mitochondrial membrane potential,[Ca～(2+)]i,reactive oxygen species were determined by flow cytometry (FCM). Results The phagocytizing activity of macrophage in VD+B group was significantly higher than that in B group 1h after infection,At the same time,the mitochondrial member potential and [Ca～(2+)]i of macrophage in VD+B group were distinctly lower than that in B group; but reactive oxygen species of macrophage in the VD+B group was insignificantly different from B group. Conclusion Vitamin D can reinforce the phagocytizing activity of macrophage and inhibit the apoptosis of macrophage after phagocytize Staphylococrcus aureus.

Objective To clone express and purify Schistosoma japonicum fructose?1 6?bisphosphate aldolase SjFBPA in E. coli and observe its expression in different developmental stages of S. japonicum. Methods FBPA gene was amplified from S. japonicum adult worm cDNA by using PCR. The amplified product was recombined into pET28a plasmid and inducibly expressed with IPTG in E. coli BL21. SDS?PAGE and Western blotting were employed to analyze and identify the recombinant protein SjFBPA rSjFBPA . Then rSjFBPA was purified by chromatographic purification and its purity was analyzed by SDS?PAGE. The protein concentration of rSjFBPA purified was measured by the BCA method. Furthermore SjFBPA mRNA was ana?lyzed in different developmental stages of S. japonicum by RT?PCR. Results SjFBPA was successfully amplified by using PCR and identified by restriction enzyme digestion and sequencing. The Western blotting analysis confirmed that the recombinant pro?tein could specifically reactive to the anti?His?tag monoclonal antibody. The concentration of the purified recombinant protein was about 4 mg/ml. The result of RT?PCR showed that SjFBPA mRNA was expressed in cercaria schistosomulum adult worm and egg of S. japonicum. Conclusion SjFBPA is successfully recombined and expressed in a prokaryotic system and SjFBPA mRNA is expressed in cercaria schistosomulum adult worm and egg of S. japonicum.

AIM: To investigate the role of gene poly mo rphisms of insulin receptor substrate-1 (IRS-1) in northern Chinese Han pedigree s with type 2 diabetes mellitus (DM). METHODS: Polymorphisms in codon 804 and 971 of IRS-1 gene in 80 unrelated patients with type 2 DM and 80 control subjects were analyzed by polym erase chain reaction-sequence specific primer(PCR-SSP) technique followed by pol yacrylamide gel electrophoresis and silver staining. RESULTS: The frequencies (GCA→GCG) in codon 804 of IRS-1 gene w ere significantly higher in type 2 DM than normal subjects(0.200 vs 0.062, P

Objective To evaluate the effect of hydrogen peroxide (H2O2) on autophagy in melanocytes,and to explore its possible regulatory mechanisms.Methods Normal human melanocytes at exponential growth phase were divided into several groups:blank control group receiving no treatment,positive control group treated with 100 nmol/L sirolimus solution,and experiment groups treated with H2O2 solution at different volume fractions of 10-7-10-3 respectively.After 4-hour treatment,cell counting kit-8 (CCK-8) assay and flow cytometry were performed to evaluate the cellular proliferative activity and detect apoptosis of melanocytes respectively.Acridine orange staining was performed to detect autophagosome formation,transmission electron microscopy to observe ultrastructural changes of autophagosomes,and Western blot analysis to measure the expression of autophagy-specific protein Beclin 1 and microtubuleassociated protein 1 light chain 3B (LC3B).A total of 84 autophagy-related genes were analyzed by RT2 Profiler PCR Array,so as to screen differentially expressed autophagy-related genes.Results After the treatment with H2O2 at different volume fractions of 10-3,5 × 10-4,10-4,5 × 10-5,10-5,5 × 10-6 and 10-6,experiment groups showed significantly decreased cellular proliferative activity,but significantly increased apoptosis rate compared with the blank control group (F =286.95,301.23,respectively,both P ＜ 0.05).With the increase in volume fractions of H2O2,the cellular proliferative activity was significantly gradually decreased (P ＜ 0.05),while the apoptosis rate showed an opposite trend (P ＜ 0.05),except that the 5 ×10-6 H2O2 group showed no significant differences in the apoptosis rate compared with the 10-5 H2O2 group and 10-6 H2O2 group.Acridine orange staining and electron microscopy showed autophagosome formation in the 10-5 H2O2 group,10-6 H2O2 group and positive control group.Western blot analysis revealed that Beclin1 expression and LC3B-Ⅱ/LC3B-Ⅰ ratio were significantly higher in the 10-5 H2O2 group,10-6 H2O2 group and positive control group than in the blank control group (all P ＜ 0.05).RT2 Profiler PCR Array showed significant up-regulation of ATG12,ATG3,ULK1,PIK3CG,PTEN and PIK3C3 genes and significant downregulation of EIF2AK3 gene in the 10-5 H2O2 group,10-6 H2O2 group and positive control group compared with the blank control group.In the 10-5 H2O2 group and positive control group,the mTOR gene was significantly up-regulated,and the ULK2 gene was significantly down-regulated.The 10-6 H2O2 group showed no obvious changes in the expression of mTOR gene,but significant up-regulation of AMPK and JNK1 genes.Conclusion H2O2 at volume fractions of 10-5 and 10-6 can induce autophagy in melanocytes,likely by influencing the expression of some related signaling molecules.

Objective To evaluate the efficacy of systemic glucocorticoids and intravenous immunoglobulin (IVIG)for the treatment of toxic epidermal necrolysis (TEN). Methods Clinical data on TEN inpatients treated with systemic glucocorticoids alone or in combination with IVIG were collected from the Department of Dermatology, First Affiliated Hospital of Nanjing Medical University from January 2006 to December 2012. Therapeutic outcomes were evaluated in these patients. Statistical analysis was carried out by using a multiple linear regression analysis, binary logistic regression analysis and Cox regression analysis with the SPSS 16.0 software. Results A total of 48 inpatients with TEN were included in this study. Multiple linear regression analysis showed that the maximum daily dose of glucocorticoids for disease control was decreased gradually over years (β=-0.461, P=0.004). However, binary logistic regression analysis revealed no obvious changes in the frequency of administration of IVIG over years. Cox regression analysis showed that both hospitalization duration (RR=0.351, 95.0%CI:0.150-0.825)and the time required for the control of skin lesions (RR=0.492, 95.0%CI:0.245-0.986)decreased with the increase in the frequency of IVIG administration. In addition, with the increase in the maximum daily dose of glucocorticoids for disease control, the time required for the control of skin lesions was also shortened (RR=0.997, 95.0%CI:0.994 -1.000), while no obvious changes were observed in hospitalization duration. Conclusions IVIG shows superiority in controlling lesions, reducing complications and improving the prognosis of TEN. Compared with systemic glucocorticoids, IVIG shows better therapeutic efficacy and less adverse effects, and may be preferentially selected.

Objective To observe effects of intranasal sufentanil on analgesia.Methods Eighty-two patients,who scheduled for selective thyroidectomy were randomly divided into the observed group and the control group with 41 cases in each group.In the observed group,patients received intranasal sufentanil 20 μg before the incision about 10 minutes,then administered local infiltration with 0.5% lidocaine.In the control group,patients received water for injection which was placebo instead sufentanil.By double blind method observed verbal mting scale(VRS)score.The values of NBP,HR,SpO2 and respiration rate(RR)were recorded before intranasal drugs and 5,10,20,40,60 min after intranasal drugs.The amount of local anesthetic,the size of thyroid gland,the time of operation and the satisfaction were compared between the two groups.Nausea vomiting and pruritus were also observed in order to assess the safety of intranasal sufentanil.Results All patients completed the operation successfully.The RR in the observed group decreased to(13.1±0.5),(13.8±0.6),(13.8±0.8)times/min after intranasal sufentanil 10,20,40 min,and had significant difference with those before intranasal drugs(P＜0.05),anastated after 60 min.VRS score of the observed group was(2.0±0.4)scales,less than the control group,and had significant difference when the surgeon was dissecting thyroid gland(P＜0.05).The amount of local anesthetic in the observed group was(42.5±6.9)ml,less than the control group(63.7±4.3)ml(P＜0.05).The satisfaction had significant difference between the two groups(P＜0.05).Conclusion Intranasal sufentanil for analgesia is a safe and useful technology.

Objective: To explore the evaluation of joint injury by HEAD-US-C (Hemophilic Early Arthropathy Detection with UltraSound in China, HEAD-US-C) in patients with moderate or severe hemophilia A treated with prophylaxis vs on-demand. Methods: The patients from June 2015 to July 2017 with moderate or severe hemophilia A were examined by ultrasound imaging of the elbows, knees and ankles; Meanwhile the HEAD-US-C ultrasound assessment scale and hemophilia joint health score scale 2.1 (HJHS2.1) were used to score the joint status. The correlation between the HEAD-US-C and HJHS score was performed in prophylaxis group and on-demand group patients, respectively. Results: A total of 925 cases of joint ultrasonography were conducted in 70 patients with moderate or severe hemophilia A. Among patients with moderate hemophilia, the median (IQR) of HEAD-US-C score and HJHS score in on-demand group were significantly higher than those in the prophylaxis group[1 (0, 6) vs 0.5 (0, 3) , z=0.177, P=0.046],[2 (0, 4) vs 2 (0, 3) z=0.375, P=0.007], even though there was no significant difference of the median (IQR) number of annualized target joints bleeding episodes between on-demand and prophylaxis groups[1 (0, 7) vs 1 (0, 5) , z=1.271, P=0.137]. Unlike in moderate cases, on-demand treatment group had more annualized target joints bleeding episodes than prophylaxis group among patients with severe hemophilia[3 (0, 8) vs 2 (0, 8) , z=0.780 P=0.037]. The prophylaxis group compared favorably with on-demand therapy group in terms of HEAD-US-C score[1 (0, 6) vs 4 (0, 7) , z=2.189, P=0.008], and HJHS score[2 (0, 5) , 4 (1, 6) , z=3646, P<0.001]for the severe hemophilia patients. The positive correlation between HEAD-US-C score and HJHS score was identified (P<0.05) , whether on-demand treatment or prophylaxis groups. The correlation coefficient between HEAD-US-C score and HJHS score in on-demand treatment and prophylaxis groups were 0.739 (95% CI 0.708-0.708) , 0.865 (95% CI 0.848-0.848) respectively, and 95% CI didn’t overlap (P<0.05) , indicating that the correlation coefficient in prophylaxis group had stronger correlation than that in on-demand group. Conclusions Clinical effects of prophylaxis were significantly better than those of on-demand treatment in patients with moderate or se-vere haemophilia A. HEAD-US-C scoring system could effectively evaluate joints damage in hemophilia A patients treated with on-demand or prophylaxis, companied by significantly positive correlation with HJHS clinical evaluation system, and provided objective index for clinical effect assessment.