Objective:To investigate apoptosis of spleen cells in infected mice by immunization with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg) and against challenge with Eg protoscoleces.Methods:BALB/c mice were vaccinated with the vaccine subcutaneously,intranasally,orally and intramuscularly respectively.The mice were then challenged with Eg protoscolexes at 8w of vaccination and sacrificed in 18w of infection to get spleen.Spleen cells were separated to measure apoptotic rate by FACsort with BCG and PBS served as control.Results:Apoptotic rate in the immunization group was lower than that in the control.Apoptotic rates in the oral or intramuscular group were significantly lower than that in the subcutaneous or intranasal group.Conclusion:Apoptosis of spleen cells in mice may be induced by infection with hydatid cyst,but is inhibited by immunization with rBCG-Eg95 vaccine.Oral or intramuscular vaccination may be the good regimen.

This article reviews the immunopathogenesis of cystic echinococcosis in the following seven aspects: innate immunity,establishment phase immunity,cystic phase immunity,influencing factor of CD4+ T cell polarization,cytokine function in infected host,Echinococcus granulosus infection and allergy,and immune evasion mechanism.

Objective:To investigate the influence of pCD-Em10 on immune responses in mice infected with alveolar hydatid cyst of Echinococcus multilocularis(Em).Methods:BALB/c mice were intramuscularly vaccinated with pCD-Em10 and then challenged with Em protoscoleces at the 8 w after vaccination.The mice were killed in 18th week of infection to count the rate of reduced alveolar hydatid cyst weight;The spleens were gathered to separate splenocytes,and the subsets of CD4+and CD8+T cells were measured by FACsort.The splenocytes were cultured by stimulation with EmAg or ConA,their supernatants were gathered to measure level of IL-2,IFN-?,TNF-? and IL-4 by ELISA Kits.Apoptotic rate of splenocytes was measured by FACsort.BCG or PBS injection served as control.Results:In the group of PCD-Em10 immunization,the rate of reduced alveolar hydatid cyst weight was 71.17%,CD4+ subset increased obviously,and the levels of IFN-? and TNF-? increased remarkably,but that of IL-4 decreased.Apoptotic rate in the pCD-Em10 group was lower than that in the PBS control.Conclusion:pCD-Em10 may induce a protective immune response in mice infected with alveolar hydatid cyst of Echinococcus multilocularis.

Objective To construct recombinant Bb-Em14-3-3 vaccine of Echinococcus multilocularis and to investigate expression efficiency of Em14-3-3 antigen encoding gene in Escherichia coli BL21(DE3).Methods Em14-3-3 antigen gene was amplified by PCR.Then the gene was cloned into Escherichia coli-Bifidobacteria shuttle plasmid pGEX-1? T containing gluathione-S-transferaze(GST) gene to construct pGEX-Em14-3-3.The recombinant plasmid was electroporated into Bifidobacteria bifidum(Bb) to construct rBb-Em14-3-3 vaccine.The vaccine was identified with PCR and restriction-endonuclease digestion.The expression of pGEX-Em14-3-3 was induced with isopropyl-?-D-thiogalactopyranosid(IPTG) and fusion protein Em14-3-3/GST was examined by SDS-PAGE and Western blot techniques.Results 530 bp gene of Em14-3-3 was successfully amplified by PCR and cloned into pGEX-1? T by restriction analysis.rBb-Em14-3-3 vaccine was successfully constructed by PCR and restriction-endonuclease digestion.It was demonstrated with SDS-PAGE and Western blot that the fusion protein Em14-3-3/GST was expressed in E.coli,BL21.The expression efficiency is 23%.Conclusion rBb-Em14-3-3 vaccine of Echinococcus multilocularis was successfully constructed.The plasmid pGEX-Em14-3-3 was highly expressed in E.coli in fused form with GST and this kind of protein shows specific antigenicity.

Objective To investigate the protection by immunization with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em) and against challenge of Em protoscolexes.Methods BALB/C mice were subcutaneously or intranasally vaccinated with mixed recombinant BCG vaccines respectively.Eight weeks later,the immunized mice were challenged with Em protoscolexes.On the 18th week after vaccination,the mice were killed to count the rate of reduced alveolar echinococcus weight and to examine the levels of IgG and its subclasses,IgE;the spleen cells were separated to measure subsets of CD4+ and CD8+ T cells by FACsort.Blank vector,BCG and PBS served as control.Results In the immunized groups,the rate of reduced alveolar echinococcus weight was 45.29%-76.47%;the levels of IgG,IgG2a,IgG2b and IgE increased apparently while IgG1 and IgG3 decreased remarkably;CD4+ subset and the ratio of CD4+/CD8+ increased obviously,but CD8+ subset had no change.Conclusion Intranasal vaccination with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine is a good way for immunization.IgG,IgG2a,IgG2b,IgE and CD4+ T cell subset may play an important role in protecting against challenge of Em protoscolexes.

Objective To investigate the reduction of hydatid cyst weight and change of splenocyte lymphokines in mice immunized with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg). Methods BALB/C mice were subcutaneously, intranasally, orally and intramuscularly vaccinated respectively, with BCG and PBS served as controls. The mice were challenged with Eg protoscolices 8 weeks after vaccination and sacrificed in 18 weeks after infection. The weight of hydatid cyst was measured and reduction of the weight was obtained, spleens were used to separate splenocytes which were cultured under stimulation with EgAg or ConA. The supernatant was collected to measure the level of IL-2、IFN-?、TNF- and IL-4 by ELISA. Results The hydatid cyst weight reduced by 45.77%, 18.20%, 88.05% and 92.46% respectively in the 4 immunization groups. In comparison with PBS control, the level of IL-2、IFN-?and TNF-?was (30.0?0) pg/ml, (65.0?0) pg/ml and (425.0?10.7) pg/ml respectively in the intramuscular group with a significant increase, but that of IL-4 decreased, with a value of (10.0?0) pg/ml. Conclusion The recombinant BCG-Eg95 vaccination induces Th1 response in mice against challenge infection.

Objective To dynamically observe changes of cytokines of splenocytes in mice by immunization with recombinant BCG-EmII/3 vaccine of Echinococcus multilocularis(Em).Methods BALB/C mice were intranasally vaccinated and then killed to get spleen on 0、2、4、6、8 and 10w of immunization respectively, splenocytes were isolated to culture with stimulation of EmAg or ConA,their supernatants were gathered to measure the level of IL-2,IFN-?,TNF-? and IL-4 by ELISA Kits. Results In the groups of immunization, levels of IL-2,IFN-?,TNF-? and IL-4 obviously increased on 2～6、2～6、2～10 and 8～10w respectively, reached the highest level on 4、4、8 and 10w respectively. Conclusion TH1 response was induced in mice by rBCG-EmII/3 vaccine by early immunization(2～8w).

Objective To analyze the clinical features of hepatic veno-occlusive disease (HVOD) and to explore the diagnosis criteria and treatment.Methods A total of 50 cases of HVOD administrated at The First Affiliated Hospital of College of Medicine,Zhejiang University from August 2008 to August 2011 were analyzed for etiology,clinical manifestation,laboratory examination,image,pathological features and treatment.Results Among the 50 HVOD cases,38 cases (76％)had a history of taking Gynura segetum.The main clinical manifestations were ascites,hepatomegaly,right upper quadrant pain,jaundice,and weight gain.Laboratory examinations indicated liver function injury and increased serum CA125 level.Color Doppler ultrasound showed hepatomegaly,decrease in diameter of hepatic vein and slow blood flow.The computed tomography scan suggested hepatomegaly,enhanced scan showed liver inhomogeneous enhancement at portal and delayed phase,map-like changes and markedly decrease in diameter of hepatic vein.Pathological examination suggested hepatic sinusoid congestion,central vein stenosis and vessel wall thickening.The main therapy was anticoagulant treatment.One case cured,33 cases improved,13 cases uncured and three cases died.Conclusions HVOD because of taking Gynura segetum is common in Zhejiang Province,which can be diagnosed according to medical history,clinical manifestations, and typical image characteristic.However,diagnosis of the atypical cases still needs liver biopsy.Anticoagulation therapy can achieve better efficacy.

The research is focused on the current situation and shortcomings in teaching of practical physical diagnostics.In the experimental group regular teachers are in charge of teaching and strengthening the physical examination training of students,and in the contrast group the teaching missions are distributed to different teachers in different departments,and the differences of the teaching effects between the two groups are thus discussed.The result reveals that the scores of the physical examination test in the experimental group is better than that in the contrast group,and the difference is significant(P