Objective To investigate the role of cAMP-PKA signal transduction pathway in the ischemie pleeonditioning-induced attenuation of ischemia.1eperfusion(ITR)injury in isohted rat hearts.Methods Fifty healthy adult SD rats weighing 300-350 g were anesthetized with intraperitoneal(IP)pentobarbital 50 meg/kg.Their he.arts were excised and perfused in a Langendorff apparatus with 37℃ oxygenated(95%O2-5%CO2)modified K-H solution at a constant pressure of 70 cm H2O,and randomly divided into 5 groups(n=10 each):group Ⅰ I/R;group Ⅱ ischemic preconditioning(IPC);groupⅢH89(PKA inhibitor);group Ⅳ PDTC(NF-κB inhibitor)and group Ⅴ db-cAMP.The experiment started after 10 min stabilization.The isolated hearts were tinct perfuzed for 30 min.followed by 60 min ischemia and 30 min leperfusion in group I/R(Ⅰ).Group IPC(Ⅱ)w88 subjected to 3 episodes of 5 min ischemia at 5 min intervals before I/R.Group Ⅲ and Ⅳ received 5 min perfnsion withH89 10 μmol/L and PDTC 100 μmol/L 3 times at 5 min intervals respectively before I/R.Group Ⅴ was peffnsed with db-cAMP 200 μmol/L for 30 min before I/R.Left ventricular developed pressure(LVDP)and ± dp/dtu were measured at stabilization period and 10, 20, 30 rain of reperfusion. Coronary flow (CF) was measured at stabilization period and 30 min of reperfusion and activities of LDH and creafinc kinase (CK) in the coronary effluent were determined. The myocardial specimens were obtained at 30 rain of reperfusion for determination of NF-κB-DNA binding activity (by EMSA) and expression of TNF-κ mBNA (by RT-PCR ) and p-CBEB (Ser133) (by Western blot). Results Compared with 1/R group, NF-κB-DNA binding activity and TNF-α mRNA expression were significandy decreased, ± dp/dt and CF were significandy increased, CK and LHD activities in the coronary effluent were significantly decreased in group IPC and db-cAMP (group Ⅱ , Ⅴ ) and p-CREB (Ser133) expression was significantly increased in group IPC, PDTC and db-cAMP (group Ⅱ , Ⅳ,Ⅴ ). Compared with IPC group, NF-κB-DNA binding activity and TNF-α mBNA expression were significantly increased, ± dp/dtmax, LVDP and CF were significantly decreased, CK and LDH activities were significantly increased in group H89 and PDTC (group Ⅲ, Ⅳ ) and p-CREB (Ser133) expression was significantly decreased in group H89(group Ⅲ ). Conclusion lschemic preconditioning can attenuate I/R injury in isolated hearts by inhibition of NF-κB-DNA binding activity via cAMP-PKA signal transduction pathway which reduces gene transcription of inflammatory cytokine.

Objective To investigate the pathogenic significance of antigenic epitopes and their relevant antibodies in pemphigus vulgaris (PV) by neonatal mouse model. Methods The extracellular domain 1-2 (EC1-2) fusion protein was expressed and purified by glutathione affinity chromatography on the basis of construction of recombinant EC1-2 vector, and then the New Zealand white rabbits were immunized to obtain the specific antisera. The IgG fraction was transferred into the neonatal mice passively after it was purified from the antisera. After 15-18 hours of injection, the abdomen skin and the sera of the mice were examined by light microscopy, electron microscopy, direct immunofluorescence and indirect immunofluorescence. Results In the evaluation of the study group of mice, the intraepithelial vesicle formation was observed. Electron microscopy showed that intercellular spaces were widened, desmosome split and disappeared. In immunofluorescence, the fluorescence-labeled IgG deposied between the acantholytic cells. In the control group of mice there were no pathogenic changes observed, except very weak fluorescence between intercellular spaces. Conclusion The PV mouse model established shows that the EC1-2 epitope in PVA antigen and its relevant antibodies were pathogenic, and can be used as a tool in studying the pathogenesis of PV.

Objective:To construct mouse ScFv against pemphigus vulgaris antigen (PVA) through phage displayed antibody technology Methods:Total RNA was isolated from the spleen cells of mice immunized with recombinant pemphigus vulgaris antigen (rPVA) after six weeks, and then reverse transcripted into cDNA Primers specific for mouse V H and V L were synthesized to amply the V H and V L by ploymerase chain reaction (PCR) V L gene was modified and amplified with linker primer (which contain 3′ region of V H, linker, and 5′ region of V L), the resultant gene called V L′ Purified V L′ and V H were subjected to SOE (splicing by overlap extension) ligation, and resultant ScFv gene was digested with SfiI and NotI, cloned to the identically digested pCantab 5E Library of TG1 transfected with recombinant phagemid were derived with diversity of 1 5?10 6 Results:The pemphigus vulgaris phage antibody library was constructed, and one positive TG1 clone was obtained, which can highly expresses phage displayed antibody Conclusion:Specific genetic antibody against PVA can be made from phage displayed antibody library

Objective To explore the infections of human cytomegalovirus (HCMV) in chronic hepatitis C patients and the hepatic impairment in chronic hepatitis C patients co-infected with HCMV.Methods HCMV-DNA was determined by fluorescence quantitative-PCR (FQ-PCR) in 95 patients with chronic hepatitis C (observation group) and 95 healthy controls(control group) and HCMV active infections were analyzed.HCV-RNA was determined by FQ-PCR in observation group,and the difference of HCMV-DNA positive rate between high HCV-RNA(＞ 104 copies/ml) and low HCV-RNA(≤ 104 copies/ml) was analyzed.Alanine aminotransferase (ALT),aspartate aminotransferase (AST) were determined by rate method in two groups and the hepatic impairment was analyzed.Results Twenty-five cases with positive HCMV-DNA in observation group,the positive rate was 26.3％(25/95).Five cases with positive HCMV-DNA in control group,the positive rate was 5.3％(5/95).There was significant difference between two groups for HCMV-DNA (x2 =14.29,P ＜0.01).Twenty-one cases with positive HCMV-DNA in 43 cases of high HCV-RNA patients,the positive rate was 48.8％(21/43).Four cases with positive HCMV-DNA in 52 cases of low HCV-RNA patients,the positive rate was 7.7％(4/52).There was significant difference between the two (x2 =19.90,P ＜ 0.01).ALT,AST in observation group was higher than that in control group (P ＜ 0.01).ALT,AST in chronic hepatitis C patients positive for HCMV-DNA was higher than that in chronic hepatitis C patients negative for HCMV-DNA significantly (P ＜ 0.01).Conclusions HCMV in chronic hepatitis C patients becomes active again and co-infects easily.When chronic hepatitis C patients co-infect HCMV actively,hepatic is further injured.

Objective MRI and MR hydrogen proton spectroscopy (1H-MRS) were used to detect the abnormal signal and alteration of metabolites, in order to explore the efficacy of these method in evaluating the damages of central nervous system (CNS) induced by occupational manganese exposure.Methods Eighteen workers exposed to manganese without any manganism symptoms, 12 workers with slightly chronic manganese poisoning, and 19 healthy workers were scanned using routine MRI sequence and 1H-MRS.The blood manganese concentration was also collected for each subject.On cerebral axial T1 WI,the signal intensities of ipsilateral globus pallidus and frontal white matter were measured in the visually brightest area (try to select the signal homogeneous region), and the globus pallidus index (PI) was then calculated.The 1H-MRS data was calculated to get the values of the peak height of N-acetylaspartate (NAA), choline (Cho), inositol (mI) and creatine (Cr) and the ratios of NAA/Cr, Cho/Cr, and mL/Cr were also calculated.One way ANOVA was used to compare the values of PI, NAA/Cr, Cho/Cr, mI/Cr and MnB among the three groups, and the correlations between PI and the time span of manganese exposure or blood manganese concentration were analyzed by Pearson correlation analysis.Eight workers exposed to manganese were followed up one year, and their PI , NAA/Cr before and after follow-up were compared by t test.Results Fourteen of 18 cases exposed to manganese without any manganism symptoms showed symmetrically high intensity signal on T1 WI, while the T2 WI were normal.No high signal intensity was observed on T1WI in any of the healthy workers or manganese poisoning workers.We found that the average PI in manganese exposed group (1.16 ±0.09) was significantly higher (F =24.79 ,P =0.O00)than those of the poisoning ( 1.05 ± 0.07 ) and control groups ( 1.01 ± 0.05 ).The blood manganese concentration in manganese exposed group, the poisoning group and the control group were (0.051 ±0.024), (0.047 ±0.018 ), ( 0.043 ± 0.020 ) μg/ml respectively, which was not significantly different ( F = O.623, P =0.541 ) and did not exceed the upper limit of normal reference value ( < 0.10 μg/ml ).There was a significantly correlation between PI and the time span of manganese exposure ( r = 0.67, P = 0.002 ),however, there was no correlation between PI and blood manganese concentration ( r = 0.20, P = 0.427 ).Furthermore, the NAA/Cr ratio decreased variously in the manganese poisoning group ( 1.22 ± 0.07 ) which was significantly lower( F = 4.120, P = 0.023 ) than those of the poisoning( 1.33 ± 0.13 ) and control groups ( 1.31 ±0.13).No statistical significanees were found in the ratios of Cho/Cr and mI/Cr among these three groups(P>0.05).No obvious changes of the PI and NAA/Cr were found in the 8 manganese exposed workers after 1 year follow-up.Conclusion Manganese exposure could lead to the high intensity signal on T1 WI, therefore the increased PI may be the biomarkers of central nerve system damages caused by the occupational manganese exposure.

Based on the current status of allocation, demands and utilization of medical care resources and the needs for future development in Shanghai, the overall objectives, principles, key plans of allocation of medical care resources in the 12th Five-years Plan in Shanghai and the leading role of health bureaus at all levels were discussed.

AIM: To investigate the role of autophagy in the apoptosis of human pulmonary artery endothelial cells (HPAECs) induced by cigarette smoke extract (CSE).METHODS: HPAECs were cultured routinely.HPAECs were treated with CSE at different concentrations, and the cell viability was detected by MTT assay.HPAECs were divided into control group, CSE group, 3-methyladenine (3-MA) group and 3-MA+CSE group.The autophagy was observed under fluorescence microscope with monodansylcadaverine (MDC) staining.Annexin V/propidium iodide staining and Hoechst 33342 staining were employed to detect apoptosis.In addition, the protein levels of LC3, beclin-1 and cleaved caspase-3 were determined by Western blot.RESULTS: MDC staining showed the increased production of autophagic vacuoles was observed in CSE group.The results of Western blot showed that the expression levels of autophagy-related proteins LC3 and beclin-1 were increased, while 3-MA pretreatment inhibited the expression of these proteins and the production of autophagic vacuoles.Observation with Annexin V/propidium iodide staining and Hoechst 33342 staining showed that the apoptotic rate in CSE group was significantly higher than that in control group, and pretreatment with 3-MA induced further increase in the cell apoptosis.The protein level of cleaved caspase-3 in CSE group was significantly higher than that in control group (P<0.05), and 3-MA+CSE treatment induced the further increase in the protein level of cleaved caspase-3.CONCLUSION: CSE induces autophagy and apoptosis in the HPAECs.Inhibition of autophagy promotes the apoptosis induced by CSE in HPAECs, which can be achieved through activation of caspase-3.

AIM:TostudythechangeofradiosensitivityofU251cellsaftertreatedwithsodiumdichloroacetate ( DCA) and further to explore the possible mechanism .METHODS: The U251 cells were divided into 4 groups: control group, DCA group, X-ray irradiation without DCA pretreatment ( IR) group and X-ray irradiation with DCA pretreatment ( DIR) group.MTT assay was applied to determine the cell viability .The intracellular reactive oxygen species ( ROS) were detected by DHE fluorescence .The expression level of Bcl-2 was assessed by Western blot .The percentage of apoptosis of cells was determined by flow cytometry .RESULTS:No difference between control group and DCA group in cell viability (P>0.05) was observed.However, the cell viability of both IR group and DIR group was markedly reduced compared with control group ( P<0.05).Furthermore, the viability of DIR group was significantly decreased compared with IR group ( P<0.05 ) .The percentage of ROS positive cells was obviously increased in DIR group compared with IR group (P<0.05).The expression level of Bcl-2 was sharply decreased in DIR group (P<0.05) and the percentage of apoptosis of cells was significantly elevated ( P<0.05) in DIR group compared with IR group .CONCLUSION:The better antitu-mor effect was obtained by improving the radiosensitivity through pretreating the cells with DCA , and the possible mecha-nism was down-regulation of the Bcl-2 expression by developing the intracellular ROS .

Objective To explore the clinical characteristics of IgG4-related disease (IgG4-RD) in Chinese by detailed clinicopathological and laboratory assessments.Methods The baseline features of 36 patients with biopsy-proven disease were reviewed.The diagnosis was confirmed by pathology review according to consensus diagnostic criteria and clinicopathologic correlation.Disease activity and damage were assessed by the IgG4-RD responder index (RI).Results Thirty (83.3％) of the patients were male,while six were female,and the average age of onset was 65.1 years.All of the 36 patients had active disease,in which submandibular gland,lymph nodes,retroperitoneal tissue were the most common affected organs in this group of patients.Among 36 patients,77.7％ had elevated serum IgG4 concentrations and 44.4％ had hypocomplementemia.Patients with elevated serum IgG4 had a higher RI,a greater number of organs involved (P ＜ 0.01 for all comparisons).The correlation between serum IgG4 level and RI (r=0.737,P ＜ 0.01) was stronger than IgG,ESR,CRP and serum complement levels.The incidence of hypocomplementemia in IgG4-RD patients with renal involvement was higher than that in IgG4-RD patients with other organs involvement (P ＜ 0.01).Twenty-eight patients received glucocorticoids therapy,and had lower RI and serum IgG4 concentration after therapy (P ＜ 0.05).Conclusions Both IgG4-RD RI and IgG4 concentration may be regarded as assessment markers of disease activity and therapeutic effect of IgG4-RD.The diagnosis of IgG4-RD should be supported by histopathology and clinical features.

A total of 1151 subjects were enrolled in this study.Metabolic syndrome (MS) was diagnosed according to the International Diabetes Federation (IDF) criteria.Significant differences in waist circumference,body mass index(BMI),diastolic blood pressure(DBP),systolic blood pressure(SBP),fat mass,Fat％ in different serum TSH levels were found.There were positive relation between fasting plasma glucose,DBP,SBP,and serum FT4 levels,between high density lipoprotein-cholesterol,DBP,SBP,waist circumference,fat mass,Fat％,and serum FT3 levels,even after adjustment for age and sex.Serum FT3 and FT4 levels were higher in the MS group than those in the control group.