| Objectives B cells changed into plasma cells, to observe the effects of different concentrations of MPA on the production of antibody, and the relationship between IMPDH2 and B cells activation and antibody production. Provide the experimental basis for the clinical use of MMF to inhibition of blood group antibody rebound.Methods ①CCK8 assay was used to observe the MPA inhibited the proliferation of B lymphocytes.Set of 0.3, 3, 30umol/L concentration of MPA group and DMSO control group, blank control group, determined the OD value of drug action time respectively, 24 h,36h, 48 h by the enzyme mark instrument, draw out the inhibitory rate and MPA concentration and action time diagram;②Select the optimum concentration of MPA,affect on the each B lymphocyte respectively, the morphological changes of B lymphocytes was observed after 4 days under a microscope;③ELISA methods to detect the inhibition of MPA to the production on B lymphocyte antibody. The same concentration of cells were seeded in 6 well plates, until the cell concentration reached 106, PWN stimulated B cells for 3-5 days change into plasma cells,add different concentration of MPA and bortezomib to the hole respectively for 4-6 days,then centrifugal, the cell supernatant was collected,ELISA detected the expression of Ig M, Ig G.④The detection rate of conversion of plasma cells by flow cytometry.The same concentration of cells were seeded in 6 well plates, until the cell concentration reached 106, the use of PWN stimulation of B cells into plasma cells after 3-5 days,respectively with different concentrations of MPA,and bortezomib to each of the holes, flow cytometry assay to detect the percentage of CD19(-) CD38(+) plasma cells after 4-6 days;⑤PCR method to detect of the influence of MPA on the B lymphocyte activate to the plasma cells. The same concentration of cells were seeded in 6 well plates, until the cell concentration reached 106, the use of PWN stimulation of B cells into plasma cells 3 days,then add different concentrations of MPA, bortezomib to the hole respectively, after 4-6 days, PCR detected the expression of IMPDH2 in each group cells;Results ① MPA for B cell proliferation inhibition, 0.03umol/ L of MPA inhibit not obvious, 30 umol / L, with the increase of drug concentration, B cell survival rate from 81.7%(0.3umol / L)decreased to 61.2%(30umol/L), increases over time 0.3umol/L MPA group survival rate from 81.7%(24h) fell to 64.15%(48h),30umol/L MPA group survival rate from61.2%(24h) fell to 38.79%(48h);②MPA inhibited plasma cells express IMPDH2;when the MPA concentration 30umol/L, IMPDH2 expression was significantly reduced, whereas 0.3-3umol/ L not obvious contrast with the control group, bortezomib group almost 0; ③MPA reduce CD19(-) CD38(+) plasma cell survival, 0.3umol / L MPA group survival rate was 14.5%,while 30umol/L MPA group survival rate drops to 8.2%. Bortezomib group was 11.8%;④Ig G antibodies over time comparing with the control group, 0.3umol-30umol/L(fourth day) MPA group decreased from 396.9ug/m L to 289.1ug/m L, compared with the control group 494.1ug/m L was significantly reduced, 0.3umol-30umol/L(sixth day) MPA group decreased from 986 ug / m L to 460ug/m L, compared with the control group 2205ug/m L was significantly reduced, while the low concentration of MPA produced little effect on Ig M, 0.3umol/L(fourth day) MPA group 254.6ng/m L compared with the control group 255.8ng/m L was not statistically significant, but 3umol / L and 30 umol /L compared with the control group decreased, decreased from 210.3ng/m L to 190.7ng/m L, bortezomib group 122.7ng /m L compared the control group was significantly reduced, the sixth day with MPA concentration increased, decreased Ig M generation, with statistical significance.Conclusion ①The MPA can inhibit the activation of B lymphocytes and B lymphocytes change into plasma cells;② The MPA inhibit plasma cell produce the Ig G, Ig M antibody may be by preventing B lymphocytes change into plasma cell or direct inhibiting of plasma cells. |
PDF Full Text Request