.fc¦Á / ¦¬r-mediated Complement Killing Of Human Glomerular Mesangial Cells

This dissertation contains two parts. In the first part, we explored the function of Fca/μR on human glomerular mesangial cells and the pathological roles in IgM nephropathy (IgMN). IgMN is an idiopathic glomerulonephritis with mesangial diffuse IgM deposits. The mechanism underlying the IgM deposition is not clear. Fca/μR is an immunoglobulin Fc receptor for both IgA and IgM. It is reported that Fca/μR was expressed on primary HMC but not on HMC cell line. To investigate whether Fca/μR could promote the deposition of IgM or IgM-IC on HMC, we firstly established a HMC line that was stably expressing human Fca/μR. Then we tested its binding ability with IgM and IgM-IC. After tried retrovirus, lentivirus and liposome to transfect, we finally decided to use liposome. The Fca/μR cDNA containing vector pcDNA3.1-Fca/μR was transfected into HMC. Drug selection and cloning culture were applied to set up a high expression cell line. The transfected Fca/μR expression was detected by Western blotting and laser scanning confocal microscopic analysis. Binding of IgM-IC to the Fca/μR on cell membrane was detected by flow cytometry and laser scanning confocal microscopyic analysis. Next, we tested if Fca/μR expressing HMC that bound IgM-IC could be killed by complement. Killing of cells by complement was analyzed by Trypan blue exclusion assay and MTT assay. After treated with complement, the death rate of Fca/μR-HMC that bound IgM-IC was significant higher than the control groups of wild type HMC, vector transfected cell and the Fca/μR-HMC incubated without IgM-IC. This indicated that IgM-IC bound by Fca/μR expressing HMC could mediate complement killing of the cells.The main work in the second part is to setup a method that can immortalize peripheral B cells from IgAN patients. To investigate the character of the aberrantly glycosylated IgAl and its secreting cells, adequate samples are needed. As cilinical samples are limited, it is necessary to establish EBV-immortalized cells from patients with IgAN. In this part, several immortalized B cell lines were established including IgAN, other nephropathy controls and heath controls. A sandwich ELISA method to detect IgA concentration was set up.