ABSTRACT
Objective To identify the miR-122 which regulateing GATA4 expression during the induction of rat bone marrow mesenchymal stem cells (bMSCs) differentiating into cardiomyocyte-like cells. Methods BMSCs were isolat-ed from bone marrow and induced to differentiate into cardiomyocyte-like cells using 5-azacytidine. The miR-122 which may regulate expression of GATA4 were predicted using miRanda and TargetScan softwares and identified by dual luciferase report system. The expressions of miR-122 and GATA4 were determined using q-PCR during the differentiation of bMSCs into cardiomyocyte-like cells. Results The induced cells were completely in contacted with adjoining cells and uniform in shape and aligned parallelly. Cardiac troponin I (cTnI) expression was detected by immunofluorescence cytochemistry. Using dual luciferase reporter system in vitro, miR-122 were proved to be able to effectively inhibit GATA4 expression by binding the 3′UTR of GATA4 mRNA. q-PCR results showed that the expression of miR-122 is negatively correlated with that of GATA4 mRNA transcription. Conclusion These results indicated that miR-122 regulate the expression of GATA4 during the induction of cardiomyocyte-like cells.
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Objective Delayed xenograft rejection (DXR) is a major barrier to the long-term xenograft survial.This study evaluated the interaction between human peripheral blood mononuelear cells (PBMC) and porcine endothelial cells (PEC),and the effects of new generation of rabbit antihuman leukocyte polyclonal antibody (newRALG) inhibiting xenogeneic cell-mediated immune responses.Methods newRALG was obtained from rabbits after immunization with activated lymphocytes and monoeytes.PEC were isolated from aorta,and human PBMC were isolated from peripheral blood.Co-cultures of PKH-26 labeled PEC with PBMC were established,newRALG,thymoglobulin,isotype Ig and scavenger receptor (SR) ligand poly G were added into the co-cultures.Cells were collected,then FACS analysis was carried out to detect the up-take of PEC membrane by monocytes and the expression of costimulatory molecules.Lymphocyte proliferative responses to PEC with or without antibody were evaluated by a xenogeneie mixed lymphocyte-endothelial cell reaction (xMLER).Results FACS analysis revealed that monocytes from PBMC-PEC co-cultures became positive for PKH-26 following their interaction with PKH-26 labeled PEC,indicating that they engulfed PEC membranes during activation.PKH-26 positive monocytes up-regulated the CD40 and CD80 expression.Furthermore,SR blockade with poly-G prevented PEC membrane up-take by monocytes,newRALG greatly reduced SR-mediated PEC membrane up-take.The effects of thymoglobulin in inhibiting PEC membrane uptake were limited.xMLER demonstrated strong lymphocyte proliferation in response to PEC,and lymphocyte proliferation was dramatically inhibited by newRALG but not isotype Ig at a dosmdependent manner.Conclusions Monocytes play an important role in xenogeneic immune responses.SR ligand poly G inhibits PEC membrane up-take.newRALG inhibits PEC membrane up-take by monocytes,suggesting that newRALG blocks SR.Additionally,newRALG inhibits lymphocyte proliferation in response to PEC.These results suggest that this new polyclonal preparation may thus impair the initiation of xeno-specific immune responses and prevent xenograft rejection.
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Objective To explore the expression and the role of monocyte-derived costimulatory molecuels during xenogeneic immune responses. Methods Porcine endothelial cells (PEC) were isolated from aorta, and subcultures were performed. CD4+ cells and monocytes were purified from human peripheral blood mononuclear cells (PBMC). PBMC-PEC co-cultures were established, and the cells were collected followed by staining with florescent-labeled monoclonal antibodies and analyzing by FACS. In selected experiments, monoclonal antibodies specific for CD154, CD80 and CD86 were added into PBMC-PEC co-cultures, and the effects of co-stimulatory molecule blockade in inhibiting lymphocyte proliferation in response to PEC were determined by 3H-thymidine up-take. The proliferation of CD4+ cells induced by PEC-conditioned monocytes with or without co-stimulation blockade was evaluated. Results PBMC-PEC co-incubation demonstrated dramatic lymphocyte proliferation as determined by 3H-thymidine up-take. FACS found that resting monocytes expressed only CD86 but not CD40 and CD80. CD14+ monocytes from PEC-stimulated PBMC demonstrated up-regulation of CD80 and CD40 expression. The up-regulation of CD86 was revealed. PEC-activated monocytes induced CD4+ cell proliferation while resting monocytes did not and this proliferation was inhibited by anti-CD154, anti-CD80 or anti-CD86 antibodies. Conclusions CD14+ monocytes play an important role during xenogeneie immune responses in indirect antigen presentation and co-stimulation- The interaction between monocyte-derived co-stimulatory molecules and CD4+ cell-derived CD154 and CD28 delivers secondary signal and induces CD4+ proliferation, and the co-stimulation blockade inhibits xe-nogeneic cell-mediated immune responses.
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Objective To investigate the immunological effects of thymoglobulin (RATG) on human CD4+and CD8+cells for costimulatory molecule gene expression and the production ofimmune-regulatory cytokines. Methods CD4+and CI8+T cells were isolated and purified fromnormal human peripheral blood mononuclear cells (PBMC) followed by incubation with RATG at37℃. Cells and culture supematants were collected at 24, 48, and 72 h after incubation, and analyzedby real-time quantitative polymerase chain reaction (RT-PCR) for CTLA-4, CD154, forkhead box P3(Foxp3), OX40, IFN-γ, IL-2, IL-10 and CD25 gene expression, and multiplex cytokine detectionassay for IFN-y, IL-2, IL-10, and IL-4 production. Untreated and rabbit isotype Ig-treated cells wereused as negative controls. Results RT-PCR demonstrated that RATG pre-treated CI+and CD8+cells upregulated the expression of CTLA-4, OX40, Foxp3, CD25, IFN-γ, IL-10 and IL-2 genes, anda dramatic increase of supernatant IFN-γ, IL-10, IL-2 and IL-4 was revealed 24 h after treatment asdetermined by multiplex cytokine detection assay when compared with negative controls. Theupre gulation of CTLA-4, Foxp3, OX40, IL-10 and CD25 was reduced, and a down-regulation ofCD154 and IL-2 gene expression was revealed 48 h after treatment. Cells, treated with RATG for 72h, demonstrated up-regulation of CTLA-4, Foxp3, OX40, IFN-y and CD25 gene expression, and theexpression of IL-2 and IL-10 genes was down-regulated. Additionally, supernatant IFN-γ, IL-2,IL-10 and IL-4 levels were decreased. Conclusion RATG stimulates CI4/CD8 T cells to up-regulatecostimulatory molecules and release immune regulation associated cytokines IF'N-γ, IL-2, IL-10in vitro. These results suggest that the unique effect of RATG on CD4+CD8+T cells may be animportant mechanism for its action in inducing immunoregulation, immunosuppression and transplanttolerance.
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Leptin,a polypeptide hormone coded by Ob-gene,has the functions of depressing appetite,reducing caloric intake and enhancing caloric consumption,etc.Leptin has a close relationship with kidney,for it is metabolized and eliminated via kidney.Meanwhile,leptin can also directly affect kidney functions,influencing the process of chronic renal failure.Furthermore,hyper-leptinemia is associated with anorexia,malnutrition and cardiovascular diseases.Different dialysis patterns may also play different roles in cleaning leptin.For example,common dialysis could not clear leptin,but other dialysis modes such as high permeability dialysis,hemodiafiltration,and mastic absorption hemoperfusion can alleviate hyper-leptinemia through cleaning leptin partly.For kidney transplantation patients,hyper-leptinemia may affect the immune balance which has been maintained for a long time.