| Objective:Chronic rejection is the major obstacle to long-term survival of allografts and is associated with graft endothelial cell activation and apoptosis. Recent reports have found an association between graft survival, presence of Th2 cytokine, and expression by endothelial cells of cytoplasmic "protective" moleculars that prevent apoptosis and down-regulate the inflammatory process.Method:1 The cultivation and identification of of boine aortic endothelial cells(BAECs)By collagenase perfusion method to isolate BAECs, which are then cultivated by DMEM containing fetal bovine serum. To identify the morphology of BAECs with the light microscope, electron microscope, and immunofluorescence. The activity and horome secretion of BAECs are determined by radioimmunoassay.2 Study of Th2 cytokine affects the expression of adhesive molecules on bovine aortic endothelial cells(1) BAECs were activated by TNF-a (4ng/ml) . Th2 cytokine (IL-4, IL-10, IL-13, 20ng/ml) were used to incubate BAECs for 2h before stimulation with TNF-a and then co-incubated for 6h or 18h. The expression of adhesive molecules(E-selectin and Intercellular adhesion molecule-1) on BAECs was measured by a Western blot method; (2) BAECs were activated by TNF-a (4ng/ml) . Th2 cytokine (IL-4, IL-10. IL-13, 20ng/ml) were used io incubate BAECs for 2h before stimulation with TNF-a and then co-incubated for 6h or 18h. The expression of adhesive molecules(E-selectin and Intercellular adhesion molecule-1) on BAECs was measured by a immuno-cell-chemical stain method; (3) BAECs were activated by TNF-a (4ng/ml). Th2 cytokine (2ng/ml 5ng/ml 10ng/mk 20ng/ml and 40ng/ml) were used to incubate BAECs for 2h before stimulation with TNF-a and then co-incubated for 6h or 18h. The expression of adhesive molecules(E-selectin and Intercellular adhesion molecule-1) on BAECs was measured by a cellular enzyme-linked immunoabsorbent assay(Cell-ELISA) method.3 Effect of Th2 cytokine(IL-4, IL-10, IL-13) on boine aortic endothelial cells(BAECs)apoptosisBy using cytometry technique, study the effect of TNF-a (10 ng/ml) on apoptosis of BAECs in vitro, and inhibitory effect of Th2 cytokine (IL-4, IL-10, IL-13, 20ng/ml) on apoptosis in TNF-a induced programmed cell death.4, Expression protective gene on BAECs induced by Th2 cytokineBAECs were activated by TNF-a (lOng/ml) . Th2 cytokine (IL-4, IL-10, IL-13, 20ng/ml) were used to incubate BAECs for 2h before stimulation with TNF-a and then co-incubated for 48h. Transcripts were detected by reverse transcription-PCR and quantitation was achieved by co-amplification of competing internal standard RNA.Results:1 Isolated BAECs grow to monolayer after one week by cultivation in vitro. They present polygonal and arrange like pavementing stones under the light microscope. W-P body canbe seen by the transmission electron microscope. Correlation antigen of VIII factor are positive by fluorescent stain. The concertration of 6-keto-PGF)aand ET is high in supernate.2 Pretreatment of BAECs with Th2 cytokine inhibited the expression of E-selectin and ICAM-1 on BAECs induced by TNF-a in a does-dependent manner.3 TNF-a is able to induce apoptosis of BAECs and treatment with Th2 cytokine can protect BAECs from TNF-a induced apoptosis in a time-dependent fashion.4, Exposure of BAECs to Th2 cytokine for 48h afford protection from activation and apoptosis induced by TNF-a (10ng/ml). This protection was associated with transcription of the genes A20 and hemoxgenase-1.Conculsions:1 The cultivated cells are proved to be BAECs, which present relative biological function of BAECs2 Th2 cytokine inhibits the expression of E-selectin and ICAM-1 on BAECs induced by TNF-a, which may contribute to the xenotransplant immune tolerance.3 Th2 cytokine inhibits BAECs apoptosis induced by TNF-a and Th2 cytokine protect BAECs from activation and damage induced by TNF-a, which may contribute to the xenotransplant immune tolerance.4 The role of Th2 cytokine protects BAECs from activation and damage induced by TNF-a may...
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