Research Of Foxo1- KLF2- S1P1 And Related Molecules Expression In Patients With Myasthenia Gravis

Background and ObjectiveMyasthenia Gravis(MG) is a kind of organ autoimmune disease in which acetylcholine(Ach R) antibody-mediated, cellular immunity depended and addiment participated cause on neuromuscular junction exist passing obstacle. The literature reports revealed that thymic epithelial cells of myasthenia gravis patient have common antigen, n ACh R, wtih skeletal muscle, which was able to sensitization T cells. Thus, activated B cells produced Ach R-Ab. 70-80% of MG patients will suffer thymic abnormal(thymoma, thymic enlargement and lymphoid follicles hyperplasia), Ach R subunits m RNA can be detected in the thymus. Thymic source T cells and B cells response to Ach R was better than the similar cells in peripheral blood, and prominent T cell dysfunction existed in the thymus of MG patients. The improvement of myasthenia gravis after excisioning thymus gland indicated the thymus plays an important role in the onset of MG. It is in the thymus that the mature T cells differentiated. A large number of humoral immune research has showed that n ACh R as target of myasthenia gravis was damage by n Ach R-Ab, while n Ach R-Ab response to Ach R was closely connected with T cells. T cells has played a key role in autoimmune of MG, but there are many problems to be studied with T cells in the thymus gland differentiation process. The development of T cells is a highly ordered and multi-stage process. Different stages of development rely on variety of different signals provided by thymic microenvironment. On the emigration of mature single positive thymus cells out of thymus, recent study shows that signals mediated by S1P1 play an important role in regulating themature single positive thymus cells export from thymus. We deemed S1P1 expresses on single positive thymus cells was the key molecules in the process of mature T cells from the thymus export into peripheral. This was confirmed by S1P1 gene defects in mice thymus cell differentiation and emigration experiment, but no confirm in human lymocyte. This provides a starting point for us to study the pathogenesis of myasthenia gravis. ObjectiveTo comparatively analyze the difference of the expression of S1P1、Foxo1、KLF2 on MG thymus between normal control thymus, discuss the role of Foxo1-KLF2-S1P1 in myasthenia gravis occurs, analyze their influence on mature T cells export from thymus of myasthenia gravis patients and relationship with the myasthenia gravis disease. MethodsCollecting 15 thymus tissues of myasthenia gravis patients(pathological characteristics was thymic hyperplasia) from the chest surgery of the second affiliated hospital of Zhengzhou University. Setting them as MG group; 15 thymus tissues of no autoimmune disease but congenital heart disease patients,setting them as normal control group. 1. Detecting m RNA of Foxo1、KLF2、S1P1 and CD62L、CCR7、CD69 : Total RNA extracted from the thymus tissue of MG patients and normal people were collected, then the m RNA expression of Foxo1、KLF2、S1P1 and CD62L、CCR7、CD69 in these tissues were examined by real-time PCR. The relative quantitative of MG group and normal control group by the 2- △ △ Ct methods. 2. Expression of molecule Foxo1 、 S1P1 and CD62 L 、 CCR7 、 CD69 on thymocyte : Paraffin-embedded sections of thymus from MG patients and normal people were made and stained with HE, then the protein expression and distribution of Foxo1、S1P1 and CD62L、CCR7、CD69 were checked by immunohistochemistry method in these Paraffin-embedded sections. 3. Quantitative analysis the proteins expression of Foxo1 and S1P1: Protein extracted from the thymus tissue, then protein level expression of Foxo1 and S1P1 were quantitative detected by Western blot. 4. Detection of S1 P level in serum : S1 P level in serum from MG patients group and normal control group were compared by ELISA. 5. Statistical analysis :The application of SPSS 17.0 statistical software for statistical analysis. Measurement data with mean ± standard deviation(SX ±),Two groups mean were compared with t tests. Think differences have statistical significance when P< 0.05. Results 1. The m RNA expression of Foxo1、KLF2、S1P1、CCR7、CD62L and CD69 in thymus tissue: The results of real-time PCR showed that the m RNA expression of Foxo1 、 KLF2 、 S1P1 、 CCR7 and CD69 in thymus tissue of MG increased significantly compared with those in thymus tissue of normal people(p<0.05), the m RNA expression of CD62 L was undifferentiated(p>0.05). 2. The molecule expression of Foxo1、S1P1、CD62L、CCR7 and CD69 on thymocyte: The results of immunohistochemistry showed that although Foxo1、S1P1 and CCR7, mainly distributed in the medulla area in thymus, expressed not only in the thymus tissue of MG patients but also normal people, the medulla area of thymus tissue in MG patient was expanded than that in normal people, and the protein expression of Foxo1、S1P1 and CCR7 was significantly higher in thymus tissue from MG patients than those from normal people(p<0.05). 3. Quantitative analysis of the proteins expression of Foxo1 and S1P1: The results of Western blot showed that Foxo1 and S1P1 expressed not only in the thymus tissue of MG patients but also normal control group, and the protein expression of Foxo1 and S1P1 was significantly higher in thymus tissue from MG patients than those from normal control group(p<0.05). 4. Content of S1 P in blood serum: The results of ELISA showed that concentration of S1 P were no difference in peripheral blood between MG patients and normal control people(p>0.05). ConclusionsThe expression of Foxo1 and its downstream molecules KLF2、S1P1 in MG thymus is significantly higher than the normal. High expression of Foxo1-KLF2-S1P1 in thymus could be the major reasons on unusual output of thymocyte cells in MG patients, providing experimental data for in-depth exploration the relationship between thymocyte cells and MG diseases.