The Expression Of Non-neuronal Cholinergic System In Peripheral Blood Mononuclear Cells Of Patients With Myasthenia Gravis

ObjectiveMyasthenia gravis(MG)is a common neuromuscular disease,which is a autoimmune disease mediated by autoimmune reaction,depend on cellular immunity and complement involved neuromuscular junction(NMJ)transmission barrier.The main target of antibody is acetylcholine receptor(AChR)at postsynaptic membrane of NMJ.There are a variety of serum autoantibodies in patients with myasthenia gravis,including acetylcholine receptor antibody(AChRab)accounted for about85%,the remaining 15% of the autoantibodies including muscle specific kinase antibody(MuSKab),low density lipoprotein antibody(LRP-4ab),and other autoantibodies.As the AChRab combined with AChR,then degraded the latter and appear skeletal muscle fatigue symptom after repeated active.In the previous study,MG patients with thymus hyperplasia group compared with normal control group,the expression of nicotinic acetylcholine receptor(nAChR)was decrease and the expression of non neuronal cholinergic system(NNCS)component in MG thymuswere significant differences(?1,?9,M3,?2,?2).The thymus is development organs for autoreactive T cells,mature mononuclear cells in peripheral blood of normal human exist NNCS,expression of NNCS component influenced immunity and antibody production.The purpose of this study was to observe whether the expression of NNCS in MG contact with the thymus or not,analysis the expression of NNCS in peripheral blood mononuclear cells(PBMC)of MG patients with and explore its effect on the emergence of autoantibodies.MethodTwenty healthy volunteers without smoking history(male 10 cases,female 10cases)and 30 MG patients from the Second Affiliated Hospital of Zhengzhou University and in Henan Province Institute of Medical Science(male 13 cases,female 17 cases)were selected,aged 25~57(39.4 + 10.4)years old,extract 6ml median cubital vein blood in heparin tube.MG patients do not take medication or only treat with pyridostigmine bromide,not associated with other autoimmune diseases.To obtain the consent of patients when collected,all patients signed informed consent,the hospital ethics committees.1.Reverse transcription PCR analysis the expression of mAChR subtypes(M1R,M2 R,M3R,M4 R,M5R)and nAChR subtypes(?2,?3,?4,?5,?6,?7,?9,?10,?2,?3,?4),choline acetyltransferase(ChAT)and acetylcholinesterase(AChE):extracted from the peripheral blood mononuclear cells RNA by Trizol method,determine the purity and concentration of RNA,observe the integrity of RNA agarose electrophoresis.RNA reverse transcription into cDNA via reverse transcription kit,amplificate under certain conditions in PCR instrument,the 5ul amplification agarose electrophoresis agarose gel electrophoresis analysis,with gel image analyzer;2.Real-time PCR analysis the expression of mAChR(M1R,M3 R,M5R),nAChR(?5,?7)and AChE: amplified cDNA in the last step in quantitative PCR instrument at certain conditions,made use of 2-??Ctmethod for analysis relative quantitative comparison between MG group and normal control group;3.Statistical methods:SPSS 21.0 statistical software for statistical analysis,single factor analysis of variance and T test were used to compare the difference ofthe expression of NNCS between the MG group and the control group,the difference was statistically significant with p < 0.05.Results1.RT-PCR analysis the expression of M1 R,M2R,M3 R,M4R,M5 R,?2,?3,?4,?5,?6,?7,?9,?10,?2,?3,?4,ChAT,AChE in patients with MG and control in PBMC: PBMC in both group expressed M3 R,M5R,alpha 5,alpha 7 beta 2 and AChE(all visible bands),however nAChR(alpha 3,alpha 9 beta 2)was only detected in the control group,the expression of the other subtypes and ChAT were not detected in the two groups(no obvious bands)2.Real-time PCR analysis the expression of mAChR(M1R,M3 R,M5R),nAChR(?5,?7)and AChE in patients with MG and control in PBMC:M1 mAChR expression level was 0.8 times higher than that of control group(p=0.418),M3 mAChR was 0.4 times higher than that of control group(p=0.004),M5 m AChR was1.23 times of the control group(p=0.488),?5 nAChR was 0.63 times higher than control group(p=0.177),?7 nAChR was 0.81 times higher than control group(p=0.668),AChE was 3.24 times higher than control group(p=0.002).ConclusionsThe expression of nAChR(?3,?9,?2),M3 mAChR and AChE in patients with MG in PBMC were different from control,thymus which is a central immune organ,play vital role on the development of T cell,mature lymphocytes migration into lymphoid organs via peripheral blood and play immune effect,the expression of these differences may be already changed in the thymus during T cells development process,further validation of the thymus paly a key role in the pathogenesis of MG.