Objective To investigate the alteration of protein kinase C (PKC) and endothelin system in early diabetic rats, and the effect of specific PKC inhibitor on the expression of retinal endothelin-1 (ET-1). Methods The rats model with streptozotocin(STZ)-induced diabetes were set up. The expression of retinal PKC was detected by enzyme-linked immunoabsorbent assay (ELISA). The expression of retinal ET-1, ET-3, ET-A and ET-B receptor mRNA was determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The alteration of retinal ET-1 mRNA after intravitreal injection of PKC inhibitor GF109203X in diabetic rats was also observed. Results The activities of membranous PKC were significantly increased in 2-week diabetic rats compared with that in normal rats(t=3.296, P=0 008), while activities of cytosolic PKC were unchangeable(t=0 138, P=0 894). The expression of retinal ET-1 mRNA was significantly increased(P=0 008), while no change was found in expression of ET-3, ET-A and ET-B mRNA(P=0 918,P=0 889,P=0 500). After intravitreal injection of 10 -5、10 -6、10 -7 mol/L PKC inhibitor GF109203X in diabetic rats, the expression of retinal ET-1 mRNA was decreased in a dose-dependent manner compared with the control rats. Conclusion Activation of PKC and increased expression of ET-1 could be found in the retina of early diabetic rats, and PKC inhibitor could inhibit the expression of retinal ET-1.

Objective To investigate the effects of transforming growth factorβ1(TGFβ1)and β3 (TGFβ3)gene transfer on MMP-2,MMP-9 and TIMP-1 expression in hepatic stellate cells(HSC-T6).Methods TGFβ1 and TGFβ3 expression plagmids were constructed.The recombinant expression plasmid pcDNA3.1(+)-=TGFβ1 and pcDNA3.1(+).TGFβ3 were transfected or cotransfected into HSC-T6.At 24,48 and 72 h after transfection,the expression of MMP-2,MMP-9 and TIMP-1 mRNA were detected by real-time quantitative PCR,and the expression of MMP-2,MMP-9 and TIMP-1 protein were detected by Western blot.The recombinant expression plasmid pcDNA3.1(+).TGFβ1 was transfected into HSC-T6,and positive clones were selected by G418.The positive clones were transfected by the recombinant expression plasmid pcDNA3.1(+).TGFβ1,and the expression of MMP-2,MMP-9 and TIMP-1 were detected at 48 h after transfection.Results After transfection with peDNA3.1-TGFβ1,MMP-2 and TIMP-1 increaged remarkably in HSC-T6 cells(P＜0.05),but MMP-9 remained at the sanle level;After transfection with pcDNA3.1-TGFβ3,expression levels of MMP-2,MMP-9 and TIMP-1 mRNA were not changed,but TIMP-1 protein increased remarkably(P＜0.05);in cotransfection group,the expression of MMP-2 was higher than that in the blank and the control groups(P＜0.05),but MMP-9 level was not changed and TIMP-1was decreased compared with that in the TGF-β1 transfection group(P＜0.05).After TGFβ3was transfected into positive clones,the change of MMP-2 wag not significant(P＞0.05).but MMP-9 increaged and TIMP-1 decreased significantly at 48 h after transfection(P＜0.05).Conclusions TGFB3 may inhibit liver fibrosis by increase the activity of MMP-2 and MMP-9,and decrease the activity of TIMP-1.

A persistent infection model was established after human hepatoma cells infected by Japanese encephalitis viruses were subcultured for several times. Viral titers of mutant viruses in persistently infected cells were examined by plaque methods using BHK cells. Nucleotides of the E coding region of two wild and two mutant viruses were amplified by RT-PCR. PCR products were sequenced by ABI-PRSM 310 sequencing system. Compared to JaGAr-01 wild strains, four amino acids were replaced (E61Tyr --> Asp, E219His --> Tyr, E384Val --> Glu, E418Pro --> Ala) in the E sequence of JaGAr-01 persistently-infected mutant strains. Eleven amino acid replacement (E51Arg --> Ser, E61Tyr --> Asp, E83Lys --> Glu, E123Ser --> Arg, E209Arg --> Lys, E227Pro --> Ser, E276Asp --> Ser, E290Arg --> Lys, E387Lys --> Arg, E418Leu --> Pro, E454Arg --> Gly) was also noted when we compared the E sequence between persistently infected Nakayama and its wild strains. A lot of similarities of amino acid sequence between mutant strains JaGAr-01 and Nakayama were also noted. It was concluded that geno-variation existed in E region of mutant viruses and the mutant protein encoded by E region, especially the mutation of E61 (Tyr --> Asp) may contribute to the maintenance of the persistent infection of Japanese encephalitis virus.

Objective To evaluate the sensitivity and specificity of new genevation Beckman Coulter cTnI in diagnosis of non-ST-elevation myocardial infarction(NSTEMI),and the values of cTnI in prognosis of non-ST elevation acute coronary syndromes(NSTEACS).Methods The cTnI was determined in NSTEMI patients and in control group,then analyzed the data with receiver operating characteristic curve (ROC curve)statistical software.Two hundred and sixty-nine patients with non-ST elevation acute coronary syndromes were divided into cTnI≥0.04 ?g/L and cTnI

Objective To explore the potential mechanisms of carcinogenesis for human eukaryotic translation elongation factor 1 alpha 2(EEF1A2).Methods Specific inhibition of EEF1A2 with siRNA was achieved in human pancreatic cancer cell line,BxPC-3,which usually expresses high level of EEF1A2.The changes of EEF1A2 expression were determined by Western blot.The effect of siRNA in suppressing the proliferation of BxPC-3 cells was determined by MTT assay,and its role in inducing BxPC-3 cell apoptosis evaluated by flow cytometry,TUNEL and transmission electron micro- scope.Results The sequence-specific siRNA effectively suppressed the expression of both EEF1A2 mRNA and protein.Specific inhibition of EEF1A2 with siRNA in pancreatic cancer cell line BxPC-3 could suppress proliferation and induce apoptosis.Conclusion The oncogenicity of EEF1A2 may be related to its role in suppressing the apoptosis and promoting the growth of pancreatic cancer cells.

Objective To evaluate the diagnostic value of endoscopic ultrasound guided fine needle aspiration (EUS-FNA) in combination with flow cytometry (FCM) in lymphoma.Methods From January 2011 to December 2011,the cases of suspicious lymphoma with EUS-FNA examination at Shanghai Ruijin Hospital were retrospectively analyzed.The final diagnosis was according to pathological diagnosis of specimen from the surgery and follow up results.The sensitivity,specificity and accuracy of EUS-FNA combined with FCM in lymphoma diagnosis were initially analyzed.Results A total of 14 suspicious lymphoma patients were collected,eight cases were diagnosed as lymphoma by pathological examination of specimen from the surgery or.tissue from aspiration,four cases were non-lymphoma lesions and two cases still had no final diagnosis.The sensitivity and specificity of FCM alone in lymphoma diagnosis were 4/8 and 4/4 respectively.Six cases of lymphoma were detected by EUS-FNA with FCM.The sensitivity,specificity and accuracy of EUS FNA combined with FCM were 6/8,6/6 and 10/12 respectively.Conclusion EUS-FNA combined with FCM has better diagnostic value in lymphoma,especially for gastrointestinal lymphoma and those surrounding deep lesions.

Objective To study the effect of brucine on the growth of a hepatocellular carcinoma cell line in vitro. Methods Brucine was added into a liver cancer cell line of SMMC-7721 in vitro, at drug concentration of brucine from 2. 5 μg/ml to 400 μg/ml. The inhibition rate of cell growth was measured by MTT technique after the cells were cultured for 72 hours. The protein and mRNA expression of PCNA,cyclin D1 and FAS were respectively assayed with Western blotting and fluorescent quantitation RT-PCR techniques at 24, 48, 72 h. Results The inhibition rate of liver cancer cell was near 100% when the brucine concentration was at 320 μg/ml. The protein and mRNA expression of FAS were of no significant difference at 24 h vs 48 h ( seperately F = 2. 547,1. 582, all P ＞ 0. 05 ), and significant difference existed at 24 h vs 72 h( seperately F = 1. 036, 1. 137, all P ＜ 0. 05 ). The protein and mRNA expression of PCNA,Cyclin D1 were of no significant difference between various time period( seperately PCNA F = 3.612,2. 174,3.029;Cyclin D1 F=2.361,2.915,1.976,all P＞0.05). Conclusions Brucine inhibits the growth of liver cancer cells, by inducing increased apoptosis of the cells probably through FAS overexpression.

Objective To investigate the expression of tumor necrosis factor alpha(TNF-α)and its relationship with infiltrating lymphocytes in lichen planus(LP).Methods Tissue specimens were obtained from the lesions of 60 patients with LP and normal skin of 20 human controls.Immunohistochemical SP method was used to detect the expression of TNF-α,and infiltrating lymphocytes were counted in TNF-α-positive tissue sections.Results TNF-α was expressed in 72％ of the LP specimens but in none of the control specimens(P ＜ 0.01).Positive staining for TNF-α was mainly located in the membrane of prickle cells,cytoplasm or membrane of dermal infiltrating lymphocytes.The expression of TNF-α in LP was uncorrelated with age,sex or disease course(all P ＞ 0.05),but was positively correlated with infiltrating lymphocyte number (rs =0.47,P ＜ 0.01).Conclusion TNF-α seems to play a certain role in the pathogenesis of LP.

Objective To compare broth microdilution and agar dilution methods for in vitro testing of activities of fluconazole,ketoconazole and itraconazole against clinical Malassezia isolates.Methods Broth microdilution and agar dilution methods were used to determine the minimal inhibitory concentration(MIC)of fluconazole,ketoconazole and itraconazole for 27 clinical strains(5 species)of Malassezia.Results The minimal inhibitory concentration(MIC)ranges of fluconazole,ketoconazole and itraconazole were 0.25-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.125 mg/L respectively as shown by broth microdilution method,2-≥64 mg/L,≤0.03-0.5 mg/L and ≤0.03-0.25 mg/L respectively as revealed by agar dilution method.Both methods demonstrated that itraconazole possessed the strongest activity against Malassezia species,followed by ketoconazole and fluconazole.The agreement rate in MICs between the two methods was 78.8％,85.2％ and 88.9％,respectively for fluconazole,ketoconazole and itraconazole,with the intraclass correlation coefficients (ICCs)being 0.88,0.80 and 0.76 respectively.Conclusions Fluconazole,ketoconazole and itraconazole are highly active against Malassezia species in vitro,and itraconazole is the most active.Broth microdilution and agar dilution method coincide well in,and are applicable for,the antifungal susceptibility testing of Malassezia species in vitro.

Objective To explore the value of EUS in the diagnosis,differential diagnosis and follow-up of primary gastric lymphoma(PGL).Methods A total of 26 patients whose endoscopic findings were highly suspected for PGL were enrolled in this study.Initial endoscopic examination anti routine biopsy were performed in all patients.EUS were undergone in all cases and its guided large piecemeal biopsy was administered in selected patients.The results of EUS,routine biopsy and routine biopsy combined with a large piecemeal biopsy were compared.Results Compared with biopsy and histopathological results,EUS made the fight diagnosis in 23 cases with overall accuracy of 88.46%,which was significantly superior to routine endoscopy and biopsy(50.0%,P=0.006),but was slightly lower than that of routine biopsy combined with large piecemeal biopsy(88.5%vs.92.3%,P=1.000).EUS can accurately differentiate gastric malignancy from benign disease with accuracy of 100.0%(23/23).Meanwhile EUS accuracy of tumor invasion(T stage)was 100.0%(12/12)as well.Conclusion EUS is valuable in the diagnosis,differential diagnosis and follow-up of PGL.