To investigate the role of CD45 protein tyrosine phosphatase in γδ T cell development,we exa-mined whether Vγ3 dendritic epidermal T cells (DETC),a subset of γδ T cells uniquely reside in the murine epidermis were altered in the CD45-gene -deficient mice.In situ immunolabelling on epidermal sheets demonstrsted that the CD45-deficient mice had a normal density and immunophenotype of Vγ3 DETC in comparison to the wild-type control mice.RT-PCR revealed that similar levels of Vγ3 TCR mRNA were present in the epidermis of both CD45-deficient mice and wild-type controls.FCM showed no significant difference in the proportion of Vγ3 T cells in the epidermal cells between the two genotypes.In addition, the frequency of Vγ2 T cells, another subset of γδT cells in lymph nodes was normal in CD45-deficient mice.These results indicate that althouh CD45 is crucial for the development of αβΤ cells,it might be not necessary for the thymic maturation of γδ T cells including Vγ3 DETC and Vγ2 T cells.

CD59 antigen is a widely expressed cell surface glycosylphosphatidyl-inositol (GPI) anchored glycoprotein.It acts as an inhibitor to the assembly of the membrane attack complex of homologous complement,binds to CD2,and also transduces activation signals with T cells.In this report,a 396bp DNA fragment was amplified by RT-PCR method from the total RNA of Jurkat cells.The fragment was cloned into pUC18 and pUC19 plas-mids,and further sequenced by Sanger′s-dideory-mediated chain termination.The results showed that this cDNA fragment included 384bp open reading fragment and its sequence was identical to the published sequence encoding human CD59 antigen.Furthermore,the cDNA of CD59 was subcloned into retroviral vector pLXSN and transfec-ted into packaging cell line PA317 to generate stable virus-producing cell lines.Then,mouse thymotase cell line EL-4 and fibroblasts cell line NIH3T3 were infected with the virus resulting in stable expression of CD59 on the cell surface.The transfected cells were tested for their susceptibility to human complement-mediated cytolysis.It was found that the transfected cells expressing CD59 antigen were far less susceptible than the controls,indicating that the gene for CD59 can be expressed in xenotypic cells stably to confer protection against human serum complement.

To seek the optimum experiment methods of animal immunization with HCV gene and to explore the effect on antibody responses in mice immunized by pCD-HCV1 recombinant in different administration, recombinant pCD-HCV1 was constructed by technique of molecular biology and was injected into muscles of Balb/c of mice with different times, routes and dosage of inoculations as well as different treatment. The results showed that the serum antibody level reached 0.183±0.06,0.428±0.05,0.707±0.08 and 0.773±0.07(OD410 value) respectively after recombinant pCD-HCV1(100μg/mouse) were injected into mice once, twice, three times and four times. The antibody level of mice (n＝12) with four times inoculation was the highest; pCD-HCV1 was perfused into stomach orally in mice or were into mice by i.p, s.c and i.m(100μg/mouse, three times) in different routes (n＝6), and the antibody levels were 0.138±0.05, 0.178±0.07, 0.233±0.08 and 0.691±0.05 respectively; after the mice (n＝8) were inoculated with the pCD-HCV1 of different dosage(10μg, 50μg and 100μg) the antibody levels of three groups were 0.11±0.09, 0.33±0.04, and 0.700±0.07, and the results showed a significant difference (P＜0.01); Mice was injected with procaine (100μl, 0.4mg) by i.m or s.c. Then pCD-HCV1 was injected into mice and antibody levels were higher than that of mice immunized directly with recombinant pCD-HCV1 of same dosage. The results may provide a reference data deserved for screening the optimum immunization method of development HCV-DNA-based vaccine in mice model.

Objective　To study the role of bcl-2 and bax in the pathogenesis of hepatitis D. Methods　Expression of HDAg， bcl-2 and bax in liver specimens of 79 patients with hepatitis D were studied by immunohistochemistry technique. Meanwhile， the relationship between expression of HDAg and that of bcl-2／bax in liver specimens of patients were studied by double labelling technique and serial sections.Results　bcl-2 was mainly expressed in the cytoplasm of hepatocytes， bax mainly in the cytoplasm of hepatocytes and partly in the nucleus of hepatocytes， and HDAg mainly in the nucleus of hepatocytes. Most of HDAg and bax positive cells were distributed among infiltrating lymphocytes at the periportal region especially at the advancing edges of areas of “piecemeal necrosis”. Most of hepatocytes of bax positive was found to locate near to positive cells of HDAg， and there were positive correlations between degrees of bax expression and HDAg expression（P＜0.05）. Conclusions　The distribution and the expression of bax and HDAg are significantly correlated with the activity of inflammation and the severity of the liver damage， and HDV infection may induce the expression of bax in the hepatocytes，and hepatocyte apoptosis mediated by bcl-2／bax may play an important role in the liver cell injury by HDV infection.

Objective　 To investigated the possible roles of IL-18 in HBV infection, and the expression of IL-18 in the peripheral blood mononuclear cells (PBMC) of chronic hepatitis B. Methods 　In 15 cases of asymptomatic carriers, 30 cases of active and remissive phases of chronic hepatitis and 10 healthy individuals (as normal controls), IL-18 expression in PBMC were quantitatively analyzed with flow cytometric immunological method. The PBMC were separated routinely and stimulated with LPS (SIGMA) and Monensin (SIGMA) for 6 hours. Then the cells were harvested and fixed by PBS/4% paraformaldehyde. The treated cells were stored in liquid nitrogen for detection later. After immunologic stain, the expression of IL-18 in PBMC were examined by flow cytometry. Results　 ① IL-18 was the lowest in asymptomatic carriers. It was fewer in remissive phase than in normal controls (P＜0.01).There was no difference between active phase and normal controls(P=0.25).②Within the group of active phase, IL-18 was significantly different between each grade of inflammatory activity(P＜0.01),and correlated with serum ALT positively(r=0.63,P＜0.01). Conclusion　 IL-18 may relate to disease activity and the inflammation of liver.

Objective　To study the mucosa immune responses of gastric and intestinal mucosa in Balb／c mice administered orally with Hp sonicate and mucosal adjuvant（LT）．Methods　The changes of antigen specific AFC in gastric and intestinal mucosa were detected by ELISPOT assay． Results　The numbers of sIgA and IgG AFC rise significantly in PP and gastric mucosa， especially the numbers of sIgA-AFC， significant differences were observed between two immunized groups and the control． Conclusions　Locally synthesized specific sIgA antibodies contribute to immunity against gastric helicobacter infection．

Objective　To obtain mammalian cell expression vector of human CD154 gene. Methods　A 820 bp cDNA fragment was amplified by RT-PCR method from total RNA of human peripheral blood mononuclear cell（PBMC） activated with 10 ng／mL PMA and 1 μg／mL PHA for 8 hours. The fragment was cloned into pcDNA3.1（＋） plasmids.The cloned insert was identified by double digestion of the recombinant plasmid with restriction enzymes BamH Ⅰ and EcoR Ⅰ and sequenced by Sangers-dideory-mediated chain termination. Results　This cDNA fragment included 820 bp entire coding region and a part of the 3 non-coding region. The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was constructed， the sequence of the insert was identical to the published sequence encoding human CD154 antigen. Conclusion　The recombinant mammalian cell expression vector of pcDNA3.1（＋）／hCD154 was successfully constructed.

In this article， several kinds of common immunosensors and the development of their transducers are introduced． Meanwhile， some problems in the fabrication of immunosensor such as immobilization method and reproduction are discussed．

Objective　To display efficient folding hDAF on the surface of yeast. Methods　The hDAF open reading frame was cloned by PCR from DAF- pBluescript M13-（Amp＋）plasmid， then subcloned into the yeast surface displayed vector pYD1.The recombinant vector was transformed into yeast cells EBY100.Flow cytometric analysis was carried out to evaluate direct binding of anti-DAF mAbs onto the surface of yeast cells displaying DAF. Results　Three mAbs against DAF different epitopes could bind onto DAF displayed on the surface of yeast.Conclusion　Efficient folding DAF can be displayed on the surface of yeast potentially leading to the development of novel applications involving DAF yeast display.

Objective　To clarify the correlation of alpha fetoprotein （AFP） half-life （T1／2） with relapse or／and metastasis of primary hepatocellular carcinoma （PHC） after treatment. Methods　Determination of AFP content in serum with two steps　double McAb sandwich ELISA.Calculation AFP　T1／2 of the sufferers treated with intervention and radiofrequency ablation （RFA） according to T1／2=0.3 dT／log（AFP1／AFP2）. Results　When AFP T1／2＞11.5 days， the sensitivity， specificity and accuracy of predicting the relapse or metastasis of PHC after treatment were 87.5％， 89.3％， 88.5％， respectively. Intervention group was 82.35％， 83.3％， 82.75％， respectively and RFA group was 93.3％， 93.75％， 93.5％， respectively. During 200 days，there　was　very　significant difference of AFP T1／2 （P＜0.001） between non-relapsed／ non-metastasized and relapsed， metastasized and died. Conclusions　AFP T1／2 　can be used as a predictive　index for relapse， metastasis of PHC after treatment. It is not only a reliable parameter for the later stage treatment， but also a useful index for the evaluation of effect.