| Aortic dissection is defined as the separation of aortic media caused by the flow of extraluminal blood within the layers of the aortic wall, which creates an extraluminal channel called the false lumen. Aortic dissection is associated with a high degree of morbidity and mortality. The detailed mechanism of aortic dissection involved in the pathological process is not completely understood. Objectives. To investigate whether autoimmune mechanism plays role in the pathogenesis or progression of Aortic dissection and elucidate the molecular mechanisms and search the targets for novel drug designs and/or biomarkers of aortic dissection. Methods. HE staining, TCA-acetone precipitation, Lysis buffer extraction, SDS-PAGE(1-D), 2-dimentional gel electrophoresis (2-D), Western blotting, LC-MS/MS, bioinformatics, and protein purification technique were used to study the expression patterns of the proteins and antigens in NC and Aortic dissection patients, detect and identify the specific autoantigens, elucidate the mechanism of aortic dissection. Results. 1. Establishment of the protein extraction. The extractions of all proteins from tissues proceeded by two different kinds of methods named TCA-acetone precipitation and Lysis buffer. We established TCA-acetone precipitation to extract all proteins of the tissues for this method was simple, fast, economic, and had high recovery rate of proteins. 2. Identification of auto-antigens: We combined the immunology and proteomics technology to identify the autoantigens. The results showed that auto-antibodies against 158 kD aorta protein in the sera of the Aortic dissection patients might exist, because the frequency and intensity of the reactivity are significantly greater in patients than in controls. The 158 kD aorta protein was found to be filamin A (actin-binding protein-280, a kind of structural protein) with LC-ESI-MS/MS. 3. Function of auto-antibodies: The proteins of various tissues were separated by 10%SDS-PAGE and followed by Western blotting to detect the reactivity of auto-antibodies. Our results showed that the auto-antibodies against aortic proteins cross-react with skeletal muscle proteins, and not cross-react with spleen proteins. The auto-antibodies reactivity detected was not found to be aortic specific. 4. The purification of filamins: Filamins were isolated from chicken gizzard smooth muscle with two different methods. The method of (NH4)2SO4 precipitation was simple, fast, economic, and had high recovery rate of filamins. After low ionic strength extraction of homogenizing, the proteins were fractionated by (NH4)2SO4-induced precipitation and ion exchange chromatography to get to purified filamins. Using LC-ESI-MS/MS the purified proteins were identified to be filamin (Gallus gallus). 5. Detection of auto-antibodies. The Western blotting were performed to screen the reactivity of the sera from patient with aortic dissection, HBP, AMI and NC controls with filamins purified from chicken gizzard. The difference was significant at P<0.01 between Aortic dissection and without aortic dissection group. The auto-antibodies against filamins were detected in patients with aortic dissection more frequently than in patients with HBP, AMI or innormal subjects. 6. The results of Hematoxylin and eosin sections: The results of HE staining showed 73.9% aortic dissection patients had inflammatory infiltrates consisting of lymphocytes and plasma cells which were present within the adventitia. Conclusions. 1. We have established the fast and effective method to extract the all proteins of the aortic tissues. 2. We have established the method to purify the filamin from the chicken gizzard fast successfully. 3. We first combined the immunology and proteomics technology to analyze the immune proteomics of NC and aortic dissection, and identified the differences of the antigen expression patterns between NC and aortic dissection. 4. The hypothesis of an autoimmune involvement in aortic dissection was first proposed. The results suggested the possible aut...
|
PDF Full Text Request