| Objective: Molecular biological techniques were used to detect the expression of AIRE,FEZF2,FOXP3,ACHRα m RNA and protein in different thymus tissues,and to explore the relationship between AIRE,FEZF2,FOXP3,ACHR and the pathogenesis of MG.Method: 1.The patients who underwent thymectomy and were pathologically diagnosed as thymoma after operation in the Department of Thoracic surgery of the first Hospital of Jilin University from January 2016 to December 2018 were collected.The patients were divided into groups according to the diagnostic criteria of myasthenia gravis: MG+thymoma group(MG-associated thymoma group,MG-T),simple thymoma group(non-MG-associated thymoma group,NMG-T)and normal thymic group(healthy control group,HC).MG-T group was thymoma complicated with MG;NMG-T group was simple thymoma without MG and other autoimmune diseases before and after 6 months follow-up;HC was thymus tissue resected during cardiac surgery to expose pericardium(excluding acute infections).All patients have signed informed consent and have been reviewed by the Ethics Committee.2.The expression of AIRE and FEZF2 in thymus was detected by immunofluorescence technique.3.The expression of AIRE,FEZF2,FOXP3 and ACHRα m RNA in thymus was detected by RT-PCR.4.Immunohistochemical technique was used to detect the expression of AIRE,FEZF2,FOXP3 and ACHR protein in thymus of MG-T and NMG-T.5.Detection of serum Ach R-Ab in patients with MG and some patients with simple thymoma by ELISA.Results: 1.There were 17 patients in MG-T group,including 6 males and 11 females,aged 45±13 years.The pathological types of thymus were classified according to WHO’s international histological classification of thymic tumors,including 5 cases of AB type and 12 cases of B2 type.The clinical stages of MG were evaluated according to Osserman classification,including 6 cases of type I,7 cases of type IIa and 4 cases of type IIb.There were 14 patients in NMG-T group,including 6 males and 8 females,aged 52±11 years.The pathological types of thymus included AB type in 6 cases and B2 type in 8 cases.There were 12 patients in HC group,including 6 males and 6 females,aged 53±11 years.2.Immunofluorescence display:almost all AIRE and FEZF2 were expressed in thymic medulla epithelial cells(Figure 4.2、4.3).3.The comparison of AIRE m RNA in MG-T,NMG-T and HC groups(Figure 4.4A): The relative value of AIRE m RNA transcription in MG-T group was lower than that in HC group(0.387(0.0714,0.9992)vs 2.492(0.5698,3.5355),and lower than that in NMG-T group(0.387(0.0714,0.9992)vs 3.413(1.2394,5.5784));P<0.05.There was no significant difference in the relative value of AIRE m RNA transcription between NMG-T group and HC group(3.413(1.2394,5.5784)vs 2.492(0.5698,3.5355),P > 0.05).4.The comparison of FEZF2 m RNA in MG-T,NMG-T and HC groups(Figure 4.4B): The relative value of FEZF2 m RNA transcription in MG-T group was lower than that in HC group(0.012(0.0017,0.0319)vs 0.154(0.09606,0.5904),P<0.05);there was no significant difference in the relative value of FEZF2 m RNA transcription between MG-T group and NMG-T group,and between NMG-T group and HC group(0.012(0.0017,0.0319)vs 0.015(0.008,0.5146),0.015(0.008,0.5146)vs 0.154(0.0960,0.5904);P > 0.05).5.The comparison of FOXP3 m RNA in MG-T,NMG-T and HC groups(Figure 4.4C): The relative value of FOXP3 m RNA transcription in MG-T group was lower than that in HC group(0.064(0.0329,1.2322)vs 2.570(0.1391,8.8592),P<0.05);there was no significant difference in the relative value of FOXP3 m RNA transcription between MG-T group and NMG-T group,and between NMG-T group and HC group(0.064(0.0329,1.2322)vs 1.118(0.2366,2.8852),1.118(0.2366,2.8852)vs 2.570(0.1391,8.8592);P>0.05).6.The comparison of ACHRα m RNA in MG-T,NMG-T and HC groups(Figure 4.4D): The relative value of ACHRα m RNA transcription in MG-T group was lower than that in HC group(0.009(0.0012,0.0873)vs 0.146(0.0384,0.1731),P<0.05);there was no significant difference in the relative value of ACHRα m RNA transcription between MG-T group and NMG-T group,and between NMG-T group and HC group(0.009(0.0012,0.0873)vs 0.097(0.0011,4.0684),0.097(0.0011,4.0684)vs 0.146(0.0384,0.1731);P>0.05).7.Comparison of AIRE,FEZF2,FOXP3 and ACHR proteins in MG-T and NMG-T groups by immunohistochemistry(Figure 4.5): The percentage of AIRE protein,FEZF2 protein,FOXP3 protein and ACHR protein in MG-T group were significantly lower than those in NMG-T group(5.585±3.294 vs 10.464±4.977,7.372±3.795 vs 12.553±5.095,0.038±0.005 vs 0.044±0.006,14.843±4.347 vs 20.949±4.366;P<0.05).Conclusion: 1.The expression of AIRE and FEZF2 was detected in thymic medulla epithelial cells.2.The expression of AIRE and FEZF2 m RNA in thymoma of MG-T group was lower than that of HC group,and the expression of AIRE m RNA was also lower than that of NMG-T group.The expression of FEZF2 protein and AIRE protein in MG-T group was significantly lower than that in NMG-T group.It is suggested that immunomodulatory factors AIRE and FEZF2 may be related to the pathogenesis of MG.3.The expression of FOXP3 m RNA in thymoma of MG-T group was lower than that of HC group,and the expression of FOXP3 protein was lower than that of NMG-T group,suggesting that the number of FOXP3 in thymoma of MG-T patients were decreased.4.The expression of ACHRα m RNA in thymoma of MG-T group was lower than that of HC group and the expression of ACHR protein was lower than that of NMG-T group.It is suggested that the expression of ACHR in thymus tissue of MG-T patients is decreased. |
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