Fas基因与重症肌无力发病机制关系的研究

Myasthenia gravis(MG) is an autoimmune disease characterized by antibodies that mainly target the acetylcholine receptor(AChR) at the neuromuscular junction and that are present in more than 85％of MG patients．The pathogenesis of MG has not yet been discovered．Fas receptor(Fas)／Fas ligand(FasL) system is one of the major forms that mediated cell apoptosis． It shows important significance to maitain thenormal immune function，to keep self-tolerance of body and to prevent the happening of autoimmune disease．It has been reported that in autoimmune lyphoproliferative syndrom(ALPS)，the downregulation of T，B cells apoptosis after activation induces autoimmune disease because of genic mutation of Fas／FasL．Little was reported about the relationship between MG and Fas／FasL．It is efficient to use glucocorticoid on the treatment of MG in clinic． Studies showed that glucoconicoid induced the apoptosis of mouse thymocytes and thymic shrink．Our research observed the Fas expressions of thymocytes and peripherial blood lymphocytes(PBLs) of MG by FCM，and studied the effect of dexamethosone(DEX) on thymocytes and Fas expression.The object is to investigate the pathogenesis of MG and the mechanisim of DEX on clinical treatment of MG．.Methodes: Thymus and blood of 35 MG patients from the First and the Second Affiliated Hospital of Zhengzhou university were diagnosed by clinical typical syndrome，Nestigmen's test and muscle electromyography. 23 controls includeded normal thymus that were obtained from 10 young adults，and blood from 13 healthy adult volunteers，average age 15．7±10．5 years．(1) The plasma and serum were isolated and the soluble Fas（sFas） molecure were assayed by Enzemy lined immunoabsorbent（ELISA）；（2）The cell surface expressions of CD4，CD8 and Fas molecules on peripheral blood mononuclear cells(PBMC) were tested by fluorochrome-coupled monoclonal antibodies(FITC-anti- Fas ，PE-anti-CD4，Cy-anti-CD8) and cytometry analysis．(3) To study the roles of hurmons and estrogen on Fas molecular expression and thymocytes apoptosis in patients with myasthenia gravis(MG)，The thymocytes were isolated from fresh thymic tissue of patients with MG. The proliferation of thymocyes was evaluated with MTT assay. The immune mediators including CD4,CD8 and Fas were analyzed on a FACScan flow cytometer. The thymocytes apoptosis was observed by agarose gel electrophoresis and cell morphology．(4)The thymus extract was prepared from thymus of patients with MG after thymus extraction operation，and the extracted protein was assayed with SDS-polyarylamide gel electrophoresis(SDS-.PAGE)．The effects of thymus extract on the proliferation of peripheral blood mononuclear cells(PBMC) were evaluated with MTT assay，and the serum AchR antibodies were evaluated with ELISA．(5) Fas gene mutations of thymocytes was detected by reverse transcription-polymerase chain reaction(RT-PCR) and single-stranded conformation polymorphism(SSCP) analysis..Results (1) The soluble Fas（sFas） molecure in plasma and serum were not found large difference between MG patients and controls.(2) the proportion of CD4+CD8- PBMC and the rate of Fas expression in patients with MC were both 1ower in comparison with those of the controls．The Fas expression of double-positive CD4+CD8+ thymocytes in patients with MG was significantly decreased，but that of single positive CD4-CD8+ thymocytes in patients with MG increased as compared with controls.(3) Both estrogen and dexasone could inhibite the proliferation of thymocytes MG patients with the difference being significant(P<0．01) between experiment group and control groups．The cultivated cells with dexasone showed character of apoptosis and the DNA of them showed typical ladder appearance in electrophoresis．The proportion of Fas thymocytes only in dexasone groups was significantly higher(P<0．05) than contro1s． Thyroxine could accelerate the proliferation of thymocytes of MG and normal human with the difference being significant(P<0．05)between experiment groups and

用ELISA、RT－PCR、SSCP（单链多肽构象分析）、免疫组化、原位杂交和DNA测序技术，对刂⒓∥蘖颊吆托灾刂ぜ∥蘖Χ锬Ｐ徒醒芯浚粤私馄浒叵僬昂笸庵苎馨拖赴托叵傧赴鸉as mRNA/DNA有钶异常，同时通过胸腺细胞和外周血淋巴细胞培养，出Fas表镆斐Ｏ赴鄄祯５钍芴逵隖as基因表达及MG发生的关系。