ABSTRACT
Objective:To study the change of related molecular about apoptosis we reduce the expression of CD59 on acute T lymphocyte Jurkat cell lines .Methods: RNA interference (RNAi) was used to reduce the expression of CD59 gene by lentivirus,confocal was applyed to observe the transfection efficiency and the location of CD59 molecular then Real-time-PCR and Western blot were used to select the most effective group to do the rest experiment;Western blot was used to detect the change of expression about Bcl-2,Bax,Caspase-3 and Survivin;ELISA was used to investigate the expression of IL-3 and TNF-β.Results: Confocal observed each group′s transfection efficiency over 90%,CD59 molecules were mainly located in cell membrane;Real-time-PCR and Western blot showed group A had the best down-regulation efficiency;we defined RNAi-CD59-A as experimental group for subsequent experiments named RNAi-CD59;the experimental group can enhance the expression of Bax,caspase-3 (P<0.05),inhibit the expression of Bcl-2 and Survivin(P<0.05);ELISA showed that the expression of IL-3 in the down-regulation group increase(P<0.05),the expression of TNF-β decrease (P<0.05).Conclusion: Down-regulation CD59 can promote the expression of apoptosis molecular in acute leukemia Jurkat cell lines restrain the expression of proliferation molecular.
ABSTRACT
BACKGROUND:The linkage and synergistic effect of adaptor proteins can effectively regulate signal transduction of T cel s, which can form a limit or amplification cascade to realize the complex immune function of T cel s. C-terminal Src kinase (Csk)-binding protein (Cbp) is an adaptor protein, which mainly exert the negative feedback regulation of Src kinase activity. This negative feedback effect depends on Y317 of Cbp, which may be involved in the SH2 domain of Csk. OBJECTIVE:To explore the effects of high expression of Cbp on ultrastructure and related biological function of Jurkat cel s. METHODS:The virus particles were constructed with expressing enhanced green fluorescent protein (EGFP) only and Cbp-EGFP fusion protein to transfect Jurkat cel s. There were untransfected group (Jurkat group), negative control group (transfected with expression of EGFP virus only), and Cbp group (transfected with Cbp-EGFP virus). RESULTS AND CONCLUSION:Confocal microscope showed that cel transfection efficiency was more than 95%and Cbp was located on the cel membrane. Optical microscope showed after transfection with Cbp-EGFP virus, more Jurkat cel s shrunk, with poor size uniformity. Apoptosis detection showed that after transfection with Cbp-EGFP virus, the number of apoptotic and necrotic cel s was greatly increased. Cbp mRNA expression was increased, Csk expression was decreased obviously and lymphocyte-specific protein tyrosine kinase expression was increased. So, in Jurkat cel s, the high expression of Cbp can decrease the uniformity of cel s and increase the necrosis cel s, thus inhibiting the signal transduction.
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Objective:To investigate the effects of chitin on atopic dermatitis in an OVA induced AD murine model.Methods:Twenty-eight BALB/c mice were randomly divided into three groups:the normal control group (N)(8),the chitin group(E) (10) and the AD model group(M)(10).The murine model of atopic dermatitis was established through intraperitoneal injection of OVA followed by repeated epicutaneous application of OVA on mice back skin( AD model group).During the set up of AD murine model,mice of the chitin group were given intragastric gavage of 3 mg/d for 4 weeks.At the end of the experiment, the mice were sacrificed and skin lesions were biopsied for histological study.HE and O-toluidine stained paraffin sections were observed under microscope.The spleen cells were cultured and challenged with OVA and chitin,respectively,the supernatant was obtained for cytokine determination.Serum levels of total and OVA-specific IgE and total IgG2a were determined with ELISA.Results:Chitin significantly inhibited skin inflammation induced by OVA.Compared with the AD model group,the thickness of the epidermis and dermis in the chitin group were obviously decreased.The numbers of dermal infiltrated inflammatory cells,eosinophils and mast cells were significantly decreased in the chitin group compared with the AD model group ( P<0.05-0.001 ).The serum level of total IgE and OVA-specific IgE were significantly lower in the chitin group than in the AD model group(P<0.05-0.001),while the serum level of IgG2a in the chitin group was significantly higher than that of the AD model group( P<0.001).The cultured spleen cells of the chitin group produced significantly higher levels of IL-12 and IFN-γ,but lower level of IL-4 compared with those of the AD model group after OVA challenge (P<0.05).Conclusion:Chitin can inhibit the inflammation and decease the seum level of IgE in the murine AD model.The antiallergic effect of chitin might be associated with the induced production of Th1 type cytokines by mice spleen cells.
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Objective:To investigate the function of CD 59 in LAT induced T lymphocytes'proliferation and activation.Methods:Transfected LAT-GFP recombinant lentiviral vectors into Jurkat cells and established a fusion-protein stable express cell line ( Jurkat-GFP ).Junket-GFP cells were transfected with pSUPER-siCD59 plasmids by electroporetion or stimulated by anti-CD59 antibody.The cellular locations of CD 59 and LAT were observed under fluorescence microscope with the immunofluorescence cytochem -istry.The cells proliferation were measured by MTT assay.Furthermore,Western blot was used to detect the total and phosphorylation levels of several down-stream proteins after T cell activated .Results: Jurkat-GFP cells successfully transfected with pSUPER-siCD59 plasmids showed lower fluorescence staining.CD59 and LAT distributed uniformly on the cell surface before stimulated with anti-CD59 antibody and formed clusters once upon stimulation.Jurkat-GFP cells stimulated with anti-CD59 antibody showed a higher level of pro-liferation and protein phosphorylation ,compared with the others.Conclusion:CD59 contributed to LAT induced signaling transduction of T lymphocytes ,and stimulated CD59 molecule partly promoted T cell activation.