Effects Of CCL25 High-level Expression On The Function Of Thymic Epithelial Cells

Background and ObjectiveMyasthenia gravis(MG) is a T-cell dependent antibody-mediated autoimmune disease characterized by muscle weakness,which develops as a result of impaired neuromuscular transmission. MG is a chronic affliction lasting many years.Evidence suggests that frequency and recognition of myasthenia gravis is increasing.no change in the number of patients aged younger than 40 years presenting, but a substantially increased age-related frequency in those over 60,with a bias towards men. Future prevalence of the disease will be determined by the spontaneous remission rate and the fact that without treatment a further 20–30% will die within 10 years. The thymus and myasthenia gravis is closely related.The thymus tissue of patients with MG often has dysplasia and thymoma. So, thymectomy is mostly used in the treatment of MG. The pathogenesis of myasthenia gravis has heterogeneity in age,changes in thymus and distribution of muscle weakness, also related to genetic factors, but the mechanism is not yet very clear.Human Chemokine CCL25, also known as thymus-expressed Chemokine,is mainly expressed in epithelial cells and dendritic cells in the thymus. CCR9 is the only receptor of CCL25, can be expressed in thymus cells, B cells, intestinal intraepithelial lymphocytes. CCR9 expressed in precursor cells from bone marrow is important to these cells homing to thymus.Our project team found CCL25 and its receptor CCR9 expression in the thymus of MG patients was higher than that in the control group through gene microarray.CCL25 overexpression in thymic stromal cellsmay be associated with the cause of MG.This study used genetic engineering techniques to build human CCL25 Lentiviral vectors, and used the vectors to infect human thymic epithelial cells Separated from the thymus in vitro, analyzed effects of CCL25 overexpression on the TEC chemotaxis, antigen-presenting function and thymocyte differentiation,explores the function of CCL25 in the pathogenesis of myasthenia gravis.MethodsNormal TEC and MG-TEC were obtained from thymus respectively by surgical resection in patients with congenital heart disease and in patients with myasthenia gravis, separated and digested with collagenase IV. And TEC was Identified with Cytokeratin monoclonal antibody and epidermal cell adhesion molecule(Ep CAM)monoclonal antibodies for Immunohistochemistry staining, and was frozen in liquid nitrogen for later experiments. Experiments had three groups of TEC: control group,infected group and MG group. The infected group compared with the control group, the MG group compared with the control group.1. Lentiviral vectors were Constructed which have human Chemokines CCL25 gene and the infection effect of them were identification.2. Normal thymus epithelial cell(TEC) was infected by virus. Then cell total RNA in three groups of cells was collected. Expression level of m RNA of Chemokines CCL5、CCL19、CCL21、CXCL10、CXCL12,Adhesion molecules P-selectin、ICAM-1 、VCAM-1、HLA-1 and HLA-2,Notch ligands DLL1、DLL4、JAG-1、JAG-2 and IL-7 was detected by fluorescent quantitative PCR, with 2-ΔΔCT method analysising relative expression differences of the gene.3. The expression of P-selectin 、 ICAM-1 and VCAM-1: total protein of three groups of cells was collected, protein expression level of P-selectin、ICAM-1 and VCAM-1 was detected by Western blot.4. The expression of proteins of IL-7 cytokine in the cell culture supernatant in infected groups、MG group and the control group were detected : putting the TEC in24-well plates, IL-7 protein level of the cell culture supernatant was detected using ELISA.5. Statistical analysis: the results were expressed as mean ± standard deviation,using statistical software SPSS 20.0 t tests, P<0.05 means the difference has statistical significance.Results1. Construction and identification of recombinant plasmid of lentiviruses with CCL25 gene: CCL25 gene with enzyme cutting sites was double digested by Xba I and Eco RI, after agarose gel electrophoresis there was a single stripe between400bp-500 bp. p CD513B-1 plasmid was double digested, electrophoretic results showed that plasmid linearization. The recombinant plasmid p CD513B-1-CCL25 was identificated by colony PCR and sequencing, the results proved that virus recombinant Plasmid was successfully identificated.2. Lentiviruses were collected and concentrated, virus titer was detected about4x108Tu/ml.3. CCL25 m RNA、CCL25 in culture supernatant concentration and CCL25 protein were tested in infected group and normal control group by fluorescence quantitative PCR、ELISAand western-blot. Compared with the control group, CCL25 m RNA and protein levels increased significantly after infection(P<0.05).4. RT-PCR showed that compared with normal control group, CCL5、CCL19、CXCL10、P-selectin、ICAM-1、VCAM-1 and IL-7 were higher(P<0.05), CCL21、CXCL12 、 HLA-A 、 HLA-DR and notch ligands expression were not changed significantly in infected group(P>0.05); CCL5、CXCL10、P-selectin、ICAM-1、VCAM-1 and IL-7 expression was significantly increased(P<0.05), CCL19 、CCL21、CXCL12、HLA-A、HLA-DR and notch ligands do not see significant differences in MG group(P>0.05).5. Western blot detection of expression of P-selectin 、 ICAM-1 and VCAM-1: compared with the control group, P-selectin、ICAM-1 and VCAM-1 proteins were significantly higher expressed in infected and MG Group(P<0.05).6. Determination of IL-7 content in the cell culture supernatant: after cells infected by the virus for 72 hours, content of IL-7 proteins was detected by ELISA.Compared with the control group, in group and MG group, IL-7 in the culture supernatant was significantly increased(P<0.05).ConclusionsOver expression of Human CCL25 genes in thymic epithelial cells can increase secretion of IL-7, increase adhesion molecules VCAM-1, ICAM-1 and p-selectin levels, affecting antigen-presenting function of the TEC.