Glycometabolism Analysis On Transgenic Mice With High Titer Of3B4Self-reactive Natural Autoantibodies

Type1diabetes mellitus (T1DM) is an organ specific autoimmune disease in whichthe pancreatic β-cells that produce insulin are the target of immune attack. The incidenceof T1DM is increasing rapidly. Although T lymphocytes play an important role in T1DM,B lymphocytes are essential for insulitis and disease progression in the non-obese diabetic(NOD) mouse model. Studies on NOD mice which were devoid of B cells have shownthat this lymphocyte population is necessary in the diabetic autoimmune process，theimportance of B lymphocytes got more and more attention. It has been proved that actingas antigen-presenting cells was one of the most important functions of B cells in T1DM.But the role of B cells that produce autoantibodies in the pathogenesis of T1DM is largelyunclear.Our group fused B cells with myeloma cell, B cells werefrom mice which was not immuned. Hybridomas which can secrete anti-keratin autoantibodies were screened, theywere polyreactive IgM autoantibodies; Thus we succeed in constructed autoantibodytransgenic mice(3B4Tg+) by cloning the variable genes of AK auto Ab.3B4Tg+derivedfrom balb/c background were backcross to NOD male mice to produce3B4Tg+/NOD.Then we discussed the relationship between glucose tolerance and autoreactive B cellsfrom3B4transgenic mice.ObjectivesEnsure the natural autoantibody which produced by3B4transgenic mice reacted withinsulin and had high affinity, observe the reaction of3B4transgenic mice after injection ofSTZ atwhich dose could not induce T1DM, and see the islet condition of3B4transgenicmice; Determine the glucose tolerance of the transgenic Balb/c and NOD mice with highserum titer of natural autoantibody which reacted with insulin.Methods1. DNA were extracted from tails of mice, PCR was adopted to identify success Heredityof3B4tansgene.2. ELISA was utilized to detect the polyreactivity of the natural autoantibody in3B4transgenic mice, affinity to bind insulin of3B4autoantibody was detect comparedwith actin as positive control; Oral glucose tolerance test (OGTT) was performed in3B4Tg+3B4Tg-male mice of C57backgroud after injection ofSTZ at which dose couldnot induce diabetes, Hematein Eosin was used to stain pancreas tissue sections(Paraffin).3. OGTT was performed in groups of WT, NOD,3B4Tg+/NOD and3B4Tg+miceat10weeks and14weeks of age; serum total IgM and anti-insulin IgM level were measuredby ELISA; Cell suspensions prepared from the peritoneal cavity and spleen wereincubated with fluorescent antibody, FACS was employed to detect the development ofB cell.Results1.3B4gene of3B4Tg+and3B4Tg+/NOD mice were stable.2. The natural autoantibody produced by3B4transgenic mice reacted with insulin and other antigens, and had a high affinity with insulin; Although both of3B4Tg+and3B4Tg-male mice of C57backgroud did not perform diabetes after injection of STZ atwhich dose could not induce T1DM,3B4Tg+mice had a higher level of blood glucoseat30min than3B4Tg-mice; mononuclear infiltrates was observed in islet of3B4Tg+mice.3.3B4Tg+/NOD and3B4Tg+mice had abnormal glucose tolerance relative to WTcontrols at10weeks of age and14weeks of age. The level of total IgM wassignificantly increased in NOD,3B4Tg+and3B4Tg+/NOD mice compared with WTmice, the level of anti-insulin IgM was significantly increased in the3B4Tg+/NODand3B4Tg+compared with NOD. The percentage of Bl a cells in peritoneal B cellsand marginal zone B cells in splenic B cells was higher in NOD,3B4Tg+/NOD and3B4Tg+than WT.ConclusionsThe natural autoantibody produced by3B4transgenic mice reacted with insulin andhad a high affinity with insulin, mononuclear infiltrates in islet of3B4Tg+mice mightberelevant to the high susceptibility of STZ. Peripheral innate immunity B cells of thenatural autoantibody3B4transgenic mice differentiate to specific subsets and produceautoantibody, mice producing high level of natural antibody with or without NODbackground performance impaired glucose tolerance, these indicate that failure of theinnate immunity tolerance is probably one of the pathogenic factors of abnormal glucosemetabolism in Type1Diabetes mellitus (T1DM).