Effects Of ScFvCD20Coupled With AntagomiR155on EAMG Mice

Part I Consctruct of scFvCD20loading the antagomiR155and exploring its effects on C57BL/6B cells under the stimulus of torpedo AchRObjective:We are aimed to construct the single-chain variable fragment of mouse CD20coupled with the antagomiR155, the inhibitor of miR155and to investigate its effects on the acetylcholine receptors-specific auto-antibodies and cytolines secretion of B cells of C57BL/6mice.Methods:Based on the gene information of CD20in genebank, we constructed the scFvCD20with the cysteine residuein the termination; the antagomiR155complemented to the miR155were coupled with the scFvCD20Cys by the cys-9R. The B cells and T cells were isolated by immunomagnetic beads and flow cytometry was used to evaluate the purity of the isolated cells. The B cells were cultured with the FITC-antagomiR155coupled with scFvCD20anf flow cytometry was used to detect the uptake rate of antagomiR155, quantitative PCR was used to detect the expression of miR155in B cells; furthermore, T cells and B cells isolated from the mice spleen were co-cultured at the ratio of1:3, meanwhile, Torpedo AchR was added into the culture, the cultured cells were randomly divided into the blank group, experimental group and the control group; the scramble antagomiRNA was used as the control of antagomiR155, flow cytometry was used to detect the CD40, CD80and CD86expression on the surface of B cells; Western blot was used to analyzed the BAFF-R related molecules; ELISA was used to detect the AchR-specific antibodies including total IgG, IgG, IgG1, IgG2a, IgG2b, IgG2c and IgG3.Results:The results from flow cytometry demonstrate that scFvCD20/antagomiR155can be uptaken by mice B cell efficiently (52.47%), which demonstrates its accuracy. Data based on quantitative PCR show that this antagomiR155can significantly inhibit the expression of miR155of mice B cells which also demonstrates the accuracy of the antagomiR155sequence.Further study demonstrates that reduce of miR155can significantly down-regulate the level of CD80,CD86and IgM on B cell surface and inhibit the expression of BAFF-R. antagomiR155; meanwhile, the phosphorylation of NIK and the translocation of NF-κB into the nucleus are also inhibited. Finally, the production of T-AchR-specific total IgG, IgG2a and IgG2b were significantly down-regulated.Conclussions:The mouse scFvCD20antibodies were successfully constructed; coupling with the antagomiR155, the scFvCD20can significantly inhibit the expression of miR155and reduce the T-AchR-specific antibodiesproduction, which can be used for the further study of EAMG therapy by silence of miR155in vivo.Part II Effects of scFvCD20loading antagomiR155on EAMG miceObjective:To explore the molecule mechanism of scFvCD20loading antagomiR155on EAMG mice for the possible mechanism.Methods:36C57BL/6mice, SPF grade, were immunized with T-AchR to establishthe animal model of EAMG, the mice were randomly divided into CFA group (n=9) and EAMG group (n=27), EAMG group were then divided into3groups (n=9,9,9),200μg T-AchR were disovled into100μl normal saline.and then mixed with equal volume CFA into antigen emulsion. The two hindlimb footpad and both shoulder were injected subcutaneously with50μl antigen emulsion and the immunization were repeated on day30and60post first immunization. On the day70.73,76and79, scFvCD20-antagomiR155were intraperitoneally injected into the EAMG mice while scFvCD20-scramble miRNA was taken as the control, EAMG mice were investigated daily for changes of clinical scores; the vena caudalis were collected for analysis of levels of anti-AchR specific antibodies; and the proportion of subgroups of B cells were detected.Results:scFvCD20-antagomiR155could ameliorate the clinical signs of EAMG while the scFvCD20-scramble miRNA has no obvious effects on the mice. The results from ELISA analysis demonstrate that inhibition of miR155significantly reduced the total IgG and IgG2a secretion of B cells; the results from flow cytometry show that the peritoneal B cell and the marginal zone B cells were both reduced significantly.Conclussion:scFvCD20loading antagomiR155could significantly ameliorate the clinical signs of EAMG mice, miR155may play a crucial role in the pathogenesi of EAMG and the miR155could be taken as the new target for the clinical therapy of MG.Part Ⅲ Expression of miR155in the PBMCs of MG patientsObjective:To investigate the expression of miR155in MG patients and the healthy controls and explore its role in the pathogenesis of MG.Methods:Collect the peripheral veinous blood of each subjects and quantitative PCR was used to detect the expression of miR155; spearman correlation was used to analyzed the relation between the miR155expression and the clinical scores of the MG patients.Results:Expression of miR155in the PBMCs of MG patients were signigicantly higher that that of the healthy control. The spearman relation analyasis shows that expression of miR155has the positive relation with the clinical signs of the MG patients.Conclussion:Together with the results from the animal experiments, our study demonstrates that miR155play a critical role in the pathogenesis of MG.