Objective:To study cardiomyocyte apoptosis of viral myocarditis (VMC) mice,study the effect and significance of Tumor Necrosis Factor-? (TNF-?) to cardiomyocyte apoptosis in VMC.Methods:Our experiments studied cardiomyocyte apoptosis at 3、5、7、9、15、35 days after virus inoculation in VMC mice by ways of TUNEL and electron microscope.We studied the effect of TNF-? to cardiomyocyte apoptosis in VMC by the way of intervention with TNF-? and TNF-? mAb.Results:Apoptotic cardiomyocyte of VMC mice increased appaently compared with control mice.Cardiomyocyte apoptosis increased after TNF-? intervention,cardiomyocyte apoptosis reduced after TNF-? mAb intervention.Conclusion:Abnormal cardiomyocyte apoptosis was observed in VMC,cardiomyocyte apoptosis may has relation with the Pathogenesis of VMC.TNF-? can induce cardiomyocyte apoptosis in VMC.TNF-? induces cardiomyocyte apoptosis may has relation with its role in VMC.

Objective:To study the effect and clinical significance of tumor necrosis factor-?(TNF-?) in viral myocarditis(VMC).Methods:Animal model was established by serum TNF-? in VMC mice was assayed by the way of ELISA.In addition,VMC mice were treated by TNF-? mAb and rTNF-?,death rate andmyocardial change of intervention group were compared with control group.Results:Serum TNF-? in VMC mice increased apparently.Inflammatory cell infiltration and myocardial necrosis had ralations with TNF-? in VMC.Inflammatory cell infiltration and myocardial necrosis in the mice of exogenous TNF-? intervention group were more serious than simple VMC group apparently,death rate of mice was also higher than simple VMC group.Inflammatory cell infiltration and myocardial necrosis in the mice of TNF-? mAb intervention group were milder than simple VMC group apparently,death rate of mice was also lower.Conclusion:TNF-? take part in the immunoreaction and pathogenesis of VMC,it has important significance to the therapy of VMC.

Objective To study ultrastructure changes of myocardium and morphic change of cardiomyocyte apoptosis in mice with viral myocarditis(VMC).Methods Our experiment established animal model of VMC by the way of coxsackie virus B3(CVB3) inoculation,then we studied the changes and apoptosis of cardiomyocyte by ways of microscope and electron microscope.Results The myocardial changes and inflammatory cell infiltration were found by ways of microscope and electron microscope from 5 days after virus inoculation in experimental mice,the peak changes were at 7-9 days,and were almost recoverd at 35 days.Apoptotic changes and apoptotic bodies were found by eletron microscope at 7-9 days after virus inoculation in mice with VMC.Conclusions VMC can be caused in mice after CVB3 inoculation,abnormal cardiomyocyte apoptosis can be found in VMC.

Objective To explore the inhibition of different immunosuppressive drugs or immunosuppressive combination regiments on the development of de novo donor specific antibodies (dnDSA).Methods We used BABL/c mice and C57BL/6 mice alloimmunized using donor splenocytes for establishing model of producing dnDSA.After that,mice were divided into 10 groups,and were administrated with mycophenolate mofetil (MMF,300 mg· kg-1 · d-1);sirolimus (0.3 mg·kg-1 ·d-1);tacrolimus(0.9 mg· kg-1 · d-1);cyclosporine A (45 mg· kg-1 · d-1);MMF (150 mg· kg-1· d-1) + tacrolimus(0.45 mg · kg-1 · d-1);MMF (150 mg · kg-1 · d-1) + cyclosporine A (22.5 mg·kg-1 ·d-1);MMF (150 mg·kg-1 ·d-1) + sirolimus (0.15 mg·kg-1 ·d-1);sirolimus (0.15 mg· kg-1 · d-1) + tacrolimus (0.45 mg· kg-1 · d-1);sirolimus (0.15 mg· kg-1 · d-1) + cyclosporine A (22.5 mg· kg-1 · d-1);and placebo;respectively.Anti-serum was harvested and tested using flow cytometry.Results (1) Four weeks after sensitization,the dnDSA level in BALB/c and C57BL/6 group was 88.86％ and 25.58％.Comparing with control group(25.33％),there was a significant increase in BALB/c group(89.23％ versus 25.33％;P＜0.001),whereas no change was found in C57BL/6 group (25.58％ versus 25.33％;P =0.259),that we chose BABL/c mice as the model for sensitization.(2)After 4 weeks of sensitization and immunosupression,dnDSA level was 29.31％,46.33％,57.10％ and 66.35％ in MMF,sirolimus,cyclosporine A and tacrolimus monotherapy group respectively.In comparison to sensitization group,dnDSA level in all monotherapy arm reduced significantly.Intra-group analysis found MMF monotherapy had the most effective inhibition of dnDSA production,sirolimus came the second,and tacrolimus and cyelosporine A had equivalent effectiveness.dnDSA level was 31.00％,33.12％,45.18％,44.62％ and 61.60％ in MMF+ sirolimus,MMF+ tacrolimus,MMF+ cyclosporine A,sirolimus + cyclosporine A and sirolimus + tacrolimus combination treatment arm,respectively,which was much lower than that of sensitization group (P＜0.001,P＜0.001,P＜0.001,P＜0.001,P =0.015).MMF + tacrolimus and MMF+ sirolimus combination treatment had comparable effectiveness in dnDSA inhibition,which is greater than that of the rest of three combination.Conclusion Our data show that the effects of single immunosuppressive drugs on the level of dnDSA is MMF＞sirolimus＞cyclosporine A =tacrolimus,and that in combination regimens is MMF + tacrolimus =MMF + sirolimus＞MMF + cyclosporine A =sirolimus + cyclosporine A =sirolimus + tacrolimus.In conclusion,this study demonstrates that MMF+ tacrolimus combination treatment arm can minimize the development of dnDSA.But,this conclusion remains to be confirmed by future clinical studies.

Objective To identify the nanobaeteria in prostate fluid of patients with CPPS.Methods Expressed prostatic secretion(EPS)and urine specimens were collected by Meares-Stamey way from CPPS patients(n=100)and normal controls(n=100).The specimens were cultured and nanobacteria was identified by indirect immunofluoreseenee staining with rnonoelonal antibody.The morphological features were observed by using transmission electron microscopy(TEM). Results The positive rate of nanobaeteria in the EPS cdture of CPPS patients and controls were 43% and 5% respectively,with significant statistical difference(X2=39.58,P＜0.01).By TEM,the sizes of NB ranged from 100 to 500 nm and appeared eoccoid-ccccobacillary shape. Conclusion Nanobaeteria infection may exist in EPS of CPPS patients.

Aim To investigate the changes of plasticity and distribution in the lungs of asthmatic guinea pigs.Methods Guinea pigs were divided into asthma group and control group.Immunohistochemistry was used in this study to observe the changes of ERK1/2 and Elk phosphoryiation in the lungs of guinea pigs.Results There were significant changes in distribution of EPK1/2 and Elk in the lungs of asthma group.The positive cells were detected on the walls of respiratory bronchioles and alveolar ducts,especially on the smooth muscle layer and basement membrane.In addition,the numbers of positive cell were clearly increased in asthma group(P< 0.01). Conclusion There is close relationship between the EPK1/2 and Elk phosphoryiation and attack of asthma.It may be a reason for persistence and progress of asthma.

The effects of DHAA-urea,a novel dehydroabietylamine(DHAA) derivatives,on cell viability and glucose metabolism,in hypoxia and normoxia human hepatoma HepG2 cells were investigated.Hypoxia cells were achieved using DMEM containing high concentration of glucose without serum and pre-incubating of CoCl_2 (final concentration 150 μmol/L) for 24 h.The antiproliferation effect of DHAA-urea was measured by colorimetric MTT assay.The cellular ATP concentration,the lactate dehydrogenase(LDH) and glucose-6-phosphate dehydro genase (G6PD) activity were detected by their kits.It was shown that DHAA-urea markedly inhibited cell viability,cellular ATP level,LDH and G6PD activity in either aerobic or anaerobic circumstance in a dose-and time dependent manner.This suggested that DHAA-urea possibly inhibited HepG2 cells growth via the inhibition of glucolysis and glucolysis-dependent ATP depletion.DHAA-urea could be a promising candidate in the development of a novel class of agents used for human hepatocellular carcinoma.

Objective A method was established for genotyping of hepatitis B virus (HBV A-D genotype),based on the PCR-restriction fragment length polymorphism (RFLP) created by Hinf Ⅰ,Ear Ⅰ,Apo Ⅰ action on an amplified segment of the S region.Methods Clinical diagnosis research.One hundred and twenty-eight HBV S sequences obtained from GeneBank were analyzed for restriction enzyme sites that would be genotype-specific.Restriction patterns following digestion with restriction enzymes Hif Ⅰ,Ear Ⅰ,Apo Ⅰ were determined to identify A-D HBV genotypes.The method was used to detect the HBV genotype of fifty severe hepatitis patients due to chronic hepatitis B in China.Then the detection results were confirmed by direct sequencing.Results The new genotyping method was established,named simple PCR-RFLP,which could identify HBV genotypes A to D.Genotypes B,C,B/C and A or D could be determined by a single step digestion with Hif Ⅰ.Eight patients of genotype A/B/C classified by single step digestion with Hif Ⅰ were conformed as genotype B variant by further digestion and direct sequencing.Extracted randomly and diluted into different concentration,three specimens were tested for genotype of HBV repeatedly and respectively.The results were all in accord with the originals,and the lowest detection limit of HBV DNA was 7 ～ 9 IU/ml.This was particularly useful in China where genotypes B and C were predominant.Twenty-three of genotype B and ten genotype C patients were classified from these fifty severe hepatitis B patients by a single step digestion with Hif Ⅰ through the simple PCR-RFLP method.The same results were also obtained by direct sequencing of PCR products (Kappa =1.00,P =0.001).The simple PCR-RFLP method was superior to direct sequencing in detecting HBV B/C polyinfection (9 cases and 0 case; x2 =18.00,P =0.001).Conclusions Both the sensitivity and repetitiveness of Simple PCR-RFLP method are satisfactory.It is superior to direct sequencing in detecting HBV B/C polyinfection,and simple,convenient.

ObjectiveTo explore the distribution of nanobacteria in prostatic calculus and investigate its role in the formation of prostatic calculus. MethodsThe stones of 40 patients with prostatic calculus was used to isolate and culture the possible bacteria. The genomes of obtained bacteria were extracted, and the 16S rRNA was amplified by PCR followed by sequencing. ResultsThe obtained specific fragment had a 98% resemblance with 16S rRNA of nanobacteria: Score=2 480 bits (1 290), Expect=0.0; Identities=1 387/1 409 (98%), Gaps=4/1 409 (0%); Strand=Plus/Plus. ConclusionNanobacteria is proved existing in the stones of prostatic calculus patients by PCR and sequencing.

A high efficiency phenol-degrading bacterial strain PS1 was isolated from the drainage ditch of chemical laboratory of East China Institute of Technology.PS1 is a coccus,Gram negative and can live on phenol as its sole carbon and energy source.PS1 to identified as a strain of Raoultella sp.by 16S rRNA gene sequence analysis,which can degrade and tolerate more than 3500mg/L phenol.When phenol concentration is 500mg/L and 1000mg/L,PS1 can completely degrade it in 22 h and 32h,respectively.And while it is between 1500mg/L～3000mg/L,all phenol can be degraded by PS1 in 32h～50h.When phenol concentration is 2500mg/L,the phenol-degrading rate is the biggest and can reach to 78.1mg/h.The optimum growth and phenol-degrading conditions were obtained by orthogonal experiment,which are 25℃,pH6.5,glucose concentration 500mg/L and 20℃,pH7.0,glucose concentration 500mg/L,respectively.