| Summary: | 博士 === 高雄醫學大學 === 醫學研究所博士班 === 104 === Objective：Thyroid orbitopathy (TO) is a multi-system autoimmune inflammatory disease characterized by orbital congestion, ocular surface disorders, restrictive myopathy and skin lesions. The molecular and cellular processes of pathogenic mechanism of TO orbital fat tissues are not fully understood. Autoimmune reactivity to orbital antigens is thought to be involved in the development of TO. Orbital adipose/connective tissues and extraocular muscle cells are thought to be targets of autoimmune attack in the disease, and they are implicated as sources of autoantigens in TO. Several autoantigens have been proposed to be involved in the pathogenesis of TO, but the autoantigen system and the mechanism of TO would be rather complex. We designed the following experiment by applying proteomic approach to further explore the possible pathogenic mechanism of TO. In first study, a comparative proteomic analysis was conducted to investigate the importance of some differential proteins of orbital fat tissues in TO. In second study, an immuno-proteomic method was conducted to survey novel autoantigens expressed in the orbital fat tissue of TO patients.
Methods：In first study, the differentially expressed proteins were analyzed by comparing the two-dimensional gel electrophoresis (2-DE) maps of the orbital fat tissues of TO with those of normal orbital fat tissues. The 2-DE results were further verified by Western blot and immunohistochemistry. In second study, we used immuno-proteomic, ELISA and immunohistochemical staining methods to survey novel autoantigens expressed in the orbital fat tissue of TO patients.
Results：In first study, fifteen up-regulated and two down-regulated proteins in TO orbital fat tissues in comparison with the control were exhibited by 2-DE maps. The over-expressed proteins including guanine nucleotide-binding protein, isocitrate dehydrogenase (IDH), annexin A2, heat shock protein 60 (HSP 60), calreticulin (CALR), protein disulfide-isomerase A3 (PDIA3), spectrin, superoxide dismutase (SOD) and transitional endoplasmic reticulum ATPase (TER ATPase) may contribute to increased thyroid stimulating hormone receptor (TSHR) expression and cell proliferation. The identified proteins are consistent with those determined by Western blot and immunohistochemistry. In second study, six protein spots showing high reactivity with the serum from the TO patients were detected as candidate orbital autoantigens, and two of them (carbonic anhydrase 1 (CA1) and alcohol dehydrogenase 1B (ADH1B)) were further verified by ELISA and immunohistochemical staining. We found carbonic anhydrase 1 (CA1) and alcohol dehydrogenase 1B (ADH1B) could attribute target autoantigens in this autoimmune disease. We discovered anti-CA1 and anti-ADH1B antibody prevalence to be higher in patients with TO (68.57 % /51.43 %) or Graves’ disease (72 % /48%) than healthy controls respectively. Immunohistochemical staining study revealed the significantly enhanced expressions of CA1 and ADH1B in orbital fat of TO compared to healthy controls.
Conclusions：In first study, comparison of orbital fat proteins from thyroid orbitopathy with age-matched controls shows significant differences in the proteome, and up-regulations of the specific proteins in orbital fat tissues from TO are associated with biochemical mechanisms or capacities against endoplasmic reticulum stress, mitochondria dysfunction, cell proliferation as well as apoptosis in TO orbital fat tissues. In second study, We found carbonic anhydrase 1 (CA1) and alcohol dehydrogenase 1B (ADH1B) could attribute target autoantigens in this autoimmune disease. The high prevalence of these autoantibodies against CA1 and ADH1B in TO and Graves’ disease (GD) patients clarify the potential clinical role for anti-CA1 and anti-ADH1B antibodies as biomarkers for GD and TO.
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