ABSTRACT
Objective To investigate the extraction and purification methods of serum specific endothelial cell antibody of renal transplant recipients with rejection after renal transplantation.Methods Human umbilical vein endothelial cell (HUVEC)was isolated and cultured.The serum samples of the renal transplant recipients with poor renal function after renal transplantation were collected.Specific endothelial cell antibody was screened out with flow cytometry;antibodies against human leukocyte antigen (HLA)and major histocompatibility complex class Ⅰ-related chain A (MICA)were detected by Luminex platform.After the existence of specific endothelial cell antibody in the serum sample was confirmed,specific endothelial cell antibody was absorbed with HUVEC.The cell was washed and then the absorbed antibody was eluted from the cell membrane.Antibody IgG in the eluent was purified and concentrated again with Protein-A /G magnetic beads.Antibody activity in the eluent was detected by flow cytometry and the purified specific endothelial cell antibody (IgG)was identified by SDS-polyacrylamide gel (SDS-PAGE)and Western blot.Results In the serum of 386 renal transplant recipients,the serum samples of 5 renal transplant recipients with serum creatinine (Scr) >400 μmoI /L,negative anti-HLA antibody,negative anti-MICA antibody and median fluorescence intensity (MFI) >16 were selected.Purified specific endothelial cell antibody IgG showed immunoglobulin heavy chain (purity > 95%)by SDS-PAGE gel.Flow cytometry showed that the purified antibody had the feature of rebinding with the surface antigen of vascular endothelial cell.Conclusions The purification method of using human umbilical vein endothelial cell to absorb specific endothelial cell antibody in the serum of renal transplant recipients may obtain good effect.
ABSTRACT
Objective To investigate the injuries caused by cholesterol to the vascular endothelial cells (VECs). Methods Different dosage of cholesterol (6.25,12.5,25.0,50.0 mg/L) was used on human umbilical endothelial cell line,ECV304,respectively. LDH activity,nitric oxide and the nitric oxide synthetase activity in the supernatant of cell culture were detected. The concentration of MCP-1 protein in cell culture was detected by ELISA. Results As compared with the normal control cells,a significant increase of LDH activity was found in the cells treated with 50.0 mg/L cholesterol. The NO level decreased in the cells treated by 25.0 or 50.0 mg/L cholesterol. When treated by cholesterol at dose of 6.25,12.5,25.0 or 50.0 mg/L respectively,the NOS activity was greatly decreased and MCP-1 protein was significantly increased in a dose-dependent manner. Conclusion Cholesterol of high concentration could directly injure the structure and partial function of VECs.