Immunoreaction Between AChRAb Purified From Myasthenia Gravis Patients Sera And Its Effects On Intracellular Calcium Concentration Of Cultured Neurons

Myasthenia Gravis is publicly known as a neuromuscular autoimmune disease,which results from the binding of AChRAb to the muscle type nicotinic AChR and leads to ACh transmission discorder. Recent years studys showed many CNS dysfunctions and electro-physiological abnormalities were associated with Myasthenia Gravis.Some incidences suggested the probable reason of CNS involvement in a part of MG patients was die cross immunoreaction between AChRAb and neuronal nicotinic AChR, but sufficient proofs were not enough.Objective: The aim of present study is to reveal the possiblity of cross immunoreaction between AChRAb and neuronal nicotinic AChR on cell level in vitro, which induces physiological changes of neurons.Methods The purified IgG were obtained from MG patients and healthy people. Cultured TE671 cell line and neurons of SD rats and Kunming mouse . Chose TE671 cell and neurons from cerebral cortex, hippocampus and brainstem of newborn SD rats at random, divided into experimental groups and control groups to make double immuhistochemical fluorescence staining. Primary antibodies used in experimental groups are IgG from MG patients and NF-160 or P Tubulin; and primary antibodies used in control groups are IgG from healthy people and NF-160 or P Tubulin. In order to furtherly cleared wheather the binding receptors of IgG fromMG were mcotinic AChR on neurons, chose three types of neurons of SD rats at random, and divided into experimental groups and control groups to make double immuhistochemical fluorescence again. Primary antibodies used in experimental groups are IgG from MG patients and VAChT, while primary antibodies used in control groups are IgG from healthy people and VAChT. All above results were observed under fluorescence microscopy and detected the fluorescence picture elemen. Then more demonstration was done by avidin-biotin complex immunocytochemcal staining on neurons from Kunming mouse embryo. Primary antibodies used in experimental groups are IgG from MG patients, while primary antibodies used in control groups are IgG from healthy people. In order to detect wheather this immunoreaction can induce physiological changes of neurons, chose neurons from cerebral cortex of SD rats to measure the intracellular Ca2^ concentration: neurons of experimental groups were incubated with MG patients seras of several doses, neurons of control groups were incubated with healthy seras of several doses including blank control. All seras were treated with complements inactivation. Added Fluo-3-AM after 2, 4, 6, 8 hours,then after incubation, washing, observed under fluorescence microscopy and detected the fluorescence picture elemen,which reflected the intracellular Ca2+ concentration. All results were analysed statistically.Results The TE671 cells of experimental groups showed green fluorescence while those of control groups did not, this demonstrated AChRAb did existed in IgG from MG patients, but did not in IgG from healthy control, since AChRAb can bind to muscle type nAChR on TE671. Neurons of experimental groups showed double staining including green fluorescences on dentrites and neuronal membranes, those of control groups showed only one staining without green fluorescences, this demonstrated AChRAb can bind to neurons of cerebral cortex, hippocampus and brainstem.Double staining of VAChT and IgG from MG patients showed their confocal reactingpositions in the same neurons, indicated AChRAb were binding to the nAChR on acetylcholine neurons. Avidin-biotin complex immunocytochemcal staining on neurons from Kunming mouse embryo showed the same results that experimental groups did have positive immunoreactions but control groups did not. The fluorescence picture elemen detection indicated there were significant varietys between the nAChR quantity of TE671 cell and neurons but not among the three types of neurons. Detection intracellular Ca2" concentration showed neurons of experimental groups changed their shapes and had intracellular Ca" over load. The level and speed of Ca"* over load we...