Effection Of Rapamycin Inhibits Proliferation Of IM-9 And Generation Of IgM, IgG

Objectives:In this study we are going to observe effect and mechanism of Rapamycin inhibits IgM, IgG produced by human B cell. So we can provide more evidences of immunosuppressive treatment for ABO incompatibility renal transplantation.Methods:①Using ELISA to observation effect of Rapamycin inhibits IgM, IgG produced by human B cell. Same concentration of Rapamycin were plated in 6-well plates. Set blank control group、DMSO group、Rapamycin 25、50 、 100nmol/L group and bortezomib positive group. Adding PWM to stimulate B cells to produce antibodies 3 days or 5 days. Then add different concentrations of drugs respectively to react 24 h, The amount of IgM, IgG generated by each Group was measured. ②The expression of CD19, CD38 was detected by FCM. Same concentration of B cell were plated in 6-well plates. Adding PWM to stimulate B cells to produce antibodies, 3 days later, then add different concentrations of Rapamycin and bortezomib respectively to react 24 h. The amount of IgM, IgG generated by each Group was measured by FCM. ③Observation Rapamycin Inhibits Proliferation of IM-9 by CCK8. Rapamycin was added in five replicates of 12.5、25、50、100、200nmol/L for each cell population, used DMSO for positive control, and blank control group. Detection each OD of Rapamycin after 24h、48h, portraying inhibitory rate diagram which showed the relationship between concentration of Rapamycin and the role of time. ④Cell cycle and apoptosis rate detection.B cell was added in 6-well plates,such as cells concentration after reaching 109 then different concentration of Rapamycin and DMSO were added into each plate. After 24 h cells were collected,centrifugal fixation,PI staining,the cells were measured by FCM. ⑤Useing PCR to detect Rapamycinmycin’s effect with Bax and Bcl-2 gene expression.Results:①Rapamycin can inhibit Proliferation of IgG, IgM. It is more effective to IgG. Inhibition is depend on dosage of Rapamycin. ②Rapamycin can reduce the cell survival rate of plasmablast effectively. ③Rapamycin can inhibit IM-9 cells significantly in vitro, Rapamycin has the most effect when concentration is 200nmol/L and the time is 48 h. ④Rapamycin can increase G1 phase. ⑤ PCR experiment showed that each group’s BCL-2 protein concentration reduced significantly to the control group. However there is no discrepancy in Bax group.Conclusion:①Rapamycin can inhibit B cells transform to plasmablasts consequently reducing generation of IgG, IgM.②Rapa can block B cells in G1 phase, thus inhibiting proliferation of B cell. meanwhile Rapamycin is likely to promote apoptosis of B cell by reducing expression of Bcl-2.