The Influence Of The Knockdown Of Partial Genes In TAM Signaling Pathyway On Mouse Thyroid Gland Function And Morphology

Objective: This study is divided into two parts.Part 1: Autoimmune thyroiditis is a common organ-specific autoimmune disease,and its pathogenesis has not been fully elucidated.The TAM receptor signaling pathway has attracted attention for its important role in negatively regulating innate immune responses and enhancing clearance of apoptotic cells.Multiple studies have found that TAM gene knocdown is closely related to the occurrence and development of multiple autoimmune diseases such as systemic lupus erythematosus,rheumatoid arthritis,Sj?gren’s syndrome and multiple sclerosis in mice.However,its effects on mouse thyroid are unclear.This study aims to reveal the knockdown of partial genes in TAM signaling pathyway on mouse thyroid function and morphology.Part 2: Transmembrane activator and CAML interactor(TACI)is an important receptors of B cell activation factor / a proliferation-inducing ligand pathway system.In this study,the serum soluble TACI(s TACI)levels of patients with different types of autoimmune thyroid diseases(AITD)were detected,and the correlation between s TACI and thyroid function and antibodies was analyzed to explore the role of s TACI in the occurrence of AITD.Methods:Part 1: female wild-type mice,homozygous Axl single-knockdown mice and homozygous Tyro3 / Mertk double-knockdown mice with were selected as research objects,with 20 mice in each group.1.Hematoxylin-eosin(HE)staining morphology was used to analyze the level of inflammatory infiltration of thyroid tissue in mice.2.Enzyme-linked immunoabsorbant assay(ELISA)was used to detect the serum thyroglobulin antibody(Tg Ab)levels in three groups of mice.3.Flow cytometry was used to detect the level of apoptosis in mouse thyroid tissue.4.Real-time fluorescent quantitative polymerase chain reaction(PCR)method to detect messenger ribonucleotide(m RNA)relative expression of mouse TAM receptor anti-inflammatory pathway molecules and TLR pathway molecules that can be negatively regulated by TAM receptors.Part 2: Recruitment of newly diagnosed patients with Graves’ disease(GD)and Hashimoto’s thyroiditis(HT)in the outpatient clinic of the First Affiliated Hospital of China Medical University,as well as healthy individuals at the medical examination center.Venous blood was collected,serum was separated,thyroid function and thyroid antibody levels were measured by electrochemical immunoluminescence,and s TACI expression levels in serum were measured by ELISA.Results:Part 1: 1.There was no obvious inflammatory cell infiltration in the thyroid gland of homozygous Axl single-knockdown mice and homozygous Tyro3 / Mertk double-knockdown mice.2.The serum Tg Ab levels of wild mice,homozygous Axl single-knockdown mice and homozygous Tyro3 / Mertk double-knockdown mice were55.24 ± 2.38 IU / m L,52.99 ± 2.60 IU / m L,and 56.18 ± 2.58 IU / m L,respectively.Compared with wild mice,there was no significant difference in serum Tg Ab levels between homozygous Axl single-knockdown mice and homozygous Tyro3 / Mertk double-knockdown mice(all P> 0.05).3.Compared with the wild mouse group,there was no significant difference in the level of apoptosis in the thyroid gland of the homozygous Axl single-knockdown mice group and homozygous Tyro3 / Mertk double-knockdown mice group(both P> 0.05).4.Compared with wild mice,the relative m RNA expression of TLR2 in thyroid tissues of homozygous Axl single-knockdown mice increased significantly(P <0.05),the relative m RNA expression of TLR9 decreased significantly(P> 0.05),and there was no significant difference in the m RNA relative expression of the remaining pathway molecules(all P> 0.05).Compared with wild mice,there was no significant difference in the relative m RNA expression of each pathway molecule in the thyroid tissue of homozygous Tyro3 / Mertk double-knockdown mice(all P> 0.05).Part 2: There was no significant difference in age and gender in the GD group and the HT group compared with the normal control(NC)group(both P> 0.05).The serum s TACI levels of patients in GD group,HT group and NC group were 13.24(10.40-18.60)pg / m L,12.75(11.17-16.14)pg / m L,and 13.05(10.30-18.75)pg / m L.Compared with the NC group,the serum s TACI levels of patients in the GD group and the HT group were not statistically different(P = 0.812,P = 0.873);there was no statistical difference of the serum s TACI expression levels between GD and HT groups(P = 0.584).In GD group,the serum level of s TACI was not correlated with free thyroxine(FT4),free triiodothyronine(FT3),thyroid stimulating hormone(TSH),thyroid peroxidase antibody(TPOAb),Tg Ab and thyrotropin receptor antibody(TRAb)level(all P>0.05).In HT group,the serum level of s TACI was not correlated with FT4,FT3,TSH,TPOAb and Tg Ab level,either(all P>0.05).Conclusion: Part 1: homozygous Axl single-knockdown mice and homozygous Tyro3 /Mertk double-knockdown mice have no obvious inflammatory cell infiltration in the thyroid gland.Compared with wild mice,there was no statistical difference in serum Tg Ab levels and apoptosis levels in thyroid tissue,either.however,the relative m RNA expression of Toll-like receptor(TLR)2 in the thyroid tissue of homozygous Axl single-knockdown mice was significantly increased,and the relative m RNA expression of TLR9 was relatively high.There was no significant difference in the relative m RNA expression of the remaining pathway molecules and there was no significant difference in the relative m RNA expression of each pathway molecule in the thyroid tissue of homozygous Tyro3 / Mertk double-knockdown mice.The abnormal expression of TLR2 and TLR9 in the thyroid tissue of homozygous Axl single-knockdown mice indicates that the abnormal expression of TLR pathway molecules in AIT which has been confirmed before may be related to the Axl receptor in the TAM signaling pathway,and the remaining multiple negative results suggested that the TAM receptor signaling pathway may not play a crucial role in the pathogenesis of AIT.Part 2: Compared with the NC group,serum s TACI levels in patients with AITD have no significant changes,and there is no correlation with thyroid function and autoimmune antibody level,suggesting that the TACI receptor in the BAFF / APRIL pathway system is not a key factor in the development of AITD.