Investigation Of The Molecular Mechanism Of That HALR Inhibited Lymphocytes Proliferation Stimulated By ConA

PART ONE INVESTIGATION OF THE MOLECULAR MECHANISM OF THE IMMUNO-MODULATION EFFECT OF HALR USING AFFYMETRIX CHIPObjective:Augmenter of liver regeneration (ALR) is a nonspecific factor that can stimulate hepatocytes proliferation. ALR is different from hepatocyte growth factor (HGF) and is nonsensitive to heat treatment. ALR is able to inhibit T cell proliferation and IL-2 secreation induced by ConA sitmulation or by antigen pulsed APC. Identification of genes regulated by ALR might help to elucidate the molecular mechanism.Methods:1. Procaryotic expression, purification and functional characterization of ALR: Using the IPTG inducible system, we expressed hALR in E.Coli SG13009. The recombinant hALR was purified using Ni-nitrilotriacetic acid resin. The activity of the purified protein was investigated via 3H-TdR incorporation assay.2. Isolation of PBMC from healthy donor , and then isolation of total RNA from PBMC.3 Comparison of gene expression in PBMC induced with ConA alone with that in PBMC treated with both ConA and hALR using Affymetrix HG-U133A plus 2.0 chip.4 Verification of differentially expressed genes using RT-PCR5 Funcational analysis of genes regulated by hALRResults:1 hALR was expressed in SG13009 E.coli cells and purified. SDS-PAGE indicated that we obtained highly purified hALR. 3H-TdR incoperation indicated that the recombinant hALR was able to stimulate proliferation of QGY cells.2 Gene expression analysis showed that by 2-fold cut-off, 1422 genes were upregulated and 1053 genes were downregulated. by 4-fold cut-off, 257 genes were upregulated and 170 genes were downregulated. Semi-quantative RT-PCR confirmed that SMAD3 and B4GALT1 were really regulated by hALR.3 Most of these hALR regulated genes are involved in different signal transduction pathways, such as: MAPK signaling pathway,JAK-STAT signaling pathway,Wnt signaling pathway, Apoptosis signaling pathway.Conclusion: 1 Recombinant hALR was expressed and purified, the activity of the recombinant protein was assayed.2 Genes regulated by hALR was identified by comparision of gene expression pattern of PBMC treated with hALR and ConA with that of PBMC treated with ConA alone.3 Most of these hALR regulated genes are involved in cell growth, proliferation and apoptosis. These genes include: Tp53,IL-1A, FAS,JunD, Bcl2, TNFRSF10A, MAP3K2 and so on. PART TWO THE RELATIONSHIP BETWEEN MAPK SIGNAL PATHWAY AND THE PROCESS OF HALR REPRESSED PROLIFERATION OF LYMPHOCYTESObjective:Gene expression analysis indicated that hALR inhibited ConA induced lymphocytes proliferation via different signal cascades, which include MAPK signaling pathway. Here we explored the significance of MAPK pathway in the process of hALR repressed proliferation of lymphocytes. Using secretion alkaline phosphatase as reporter gene, we investigated the function of hALR on the acitivities of three transcription factors: AP-1, NFAT,SRE. Then we checked the phosphorylation status of the protein kinase upstream of the transcription factors.Methods:1 The effect of hALR on ConA induced Jurkat T cells: After 6 hours induced with ConA, Jurkat T cells were treated with different doses of hALR, the effect was evaluated by 3H-TdR incorporation assay.2 Amplification and enzyme digestion of SEAP reporter plasmids: transformation of competent E. Coli JM109 with AP-1,NFAT,SRE reporter plasmids, the amplified plasmids were purified and enzyme digested. 3 Transient transfection of reporter plasmids and detection of secretion alkaline phosphatase: JurkatT cells were transfected with pAP1-SEAP, pNFAT-SEAP, pSRE-SEAP, pTAL-SEAP (negative control), pSEAP2-control (positive control) using GeneJuice. 24 hours after transfection, ConA was added, and 30 hours after transfection, hALR was added. The activity of secretion alkaline phosphatase in the medium was assayed at 0h, 6 h, and 18 h after hALR treatment.4 Detection of phosphorylation status of JNK: 6 hours after stimulation with ConA, Jurkat T cells were treated with hALR, the phosphorylation status of JNK were assayed 0, 6 and 12 hours after hALR treatment.Results:1. Effects of different concentration of hALR on the proliferation of ConA induced Jurkat T: At the concentration between 20ug/ml and 100ug/ml, hALR inhibited the proliferation of ConA induced Jurkat T cells in a dose-dependant manner.2. Ezyme digestion confirmed the indentity of reporter plasmids3. Transisient transfection with reporter plasmids indicated that: a, ConA increased the activities of AP-1, NFAT and SRE (P<0.01);b, hALR treatment for 6h and 18h further increased the activity of AP-1; c, the activities of NFAT and SRE were not affected by hALR treatment.4. Western blotting indicated that: a, ConA stimulated the phosphorylation of JNK; b, hALR treatment for 6h and 12h, the phosphorylation was further increased.Conclusion:1. The inhibitory effect of hALR on ConA induced proliferation of Jurkat T cells was confirmed, and the best inhibitory concentration was determined.2. The identity of the reporter plasmids was confirmed by enzyme digestion.3. hALR further increased the ConA induced phosphorylation of JNK, which suggests that JNK/MAPK pathway might partially account for the proliferation-inhibiting effect of hALR.