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.

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.

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.

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.

Objective:To analyze differentially expressed genes (DEGs) and the changes of signal pathways in human retinal pigment epithelium cells (ARPE-19) under hypoxic and normoxic conditions and to explore the biological mechanism of hypoxia-induced ARPE-19 cell damage via transcriptome sequencing (RNA-seq) and bioinformatics technology.Methods:The ARPE-19 cells were divided into the hypoxia treatment group and the normoxia control group treated with 1% and 21% O 2 by volume for 8, 24, 48, 72 hours, respectively.The relative expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected with real-time fluorescent quantitative PCR at different time points.RNA-seq and bioinformatics analysis were performed at 8 hours and 24 hours after hypoxia and normoxia treatment.DEGs were screened out under the conditions of |log 2FC|≥1 and P≤0.05.Then the cluster heat map analysis, Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were also carried out.Real-time fluorescent quantitative PCR was employed at 24 hours after hypoxia to detect the relative mRNA expression of genes that might be related to hypoxia in DEGs.Cell viability kit was used to verify and compare the damage effect of hypoxia on ARPE-19 cells at different time points between the two groups. Results:The relative mRNA expression levels of VEGF at 8, 24, 48 and 72 hours after hypoxia treatment and the relative HIF-1α mRNA expression levels at 8, 24 and 48 hours after hypoxia treatment were significantly higher than those of the normoxia control group (all at P<0.05). There were large differences in the mRNA expression levels at 8-hour and 24-hour treatment between the two groups.A total of 62 significant DEGs were screened between the hypoxia treatment group and the normoxia control group after 8-hour hypoxia treatment, among which 45 genes were significantly up-regulated and 17 genes were significantly down-regulated.A total of 255 significant DEGs were screened out between the hypoxia treatment group and the normoxia control group after 24-hour hypoxia treatment, among which 228 genes were significantly up-regulated and 27 genes were significantly down-regulated.The GO functional analysis of DEGs was mainly enriched in processes such as protein degradation, nucleotide biosynthesis, and material transport.KEGG pathway analysis was mainly enriched in PI3K-Akt, cGMP-PKG, and other signaling pathways closely related to metabolism, cell cycle, cell growth, and apoptosis.The core genes HPCA, MT3 and NOS3 were found by protein-protein interaction network analysis.Real-time fluorescent quantitative PCR test results showed that after 24-hour hypoxia treatment, the mRNA expression levels of hypoxia related genes DEPP1, NPPB, PDZK1, HILPDA, TCEA3, NDRG1 and RORC in ARPE-19 cells were significantly increased and the mRNA expression levels of TFRC and NQO1 were significantly decreased (all at P<0.05). The cell morphology was normal and the growth state was good without dead cells after 8-hour and 24-hour hypoxia treatment in ARPE-19 cells.There were dead cells after 48-hour hypoxia treatment, and the number of dead cells was increased at 72 hours after hypoxia treatment. Conclusions:The PI3K-Akt and cGMP-PKG signaling pathways related to metabolism may be involved in hypoxia-induced injury of ARPE-19 cells.Core genes of HPCA, MT3 and NOS3 can be used as functional target genes and play key roles in hypoxia response of cells.