| Background Vitiligo is a skin and hair disorder characterized by circumscribed depigmented lesions due to lack of melanocytes in the respective areas. Recently, its incidence rate gradually increased. Lack of melanocytes mainly present in epidermis, melanocytes in hair follicle also are involved. However, the mechanism is still unknown utterly. At present, there are some theory about vitiligo, such as immune hypothesis, the genetic theory, autocytotoxic hypothesis and neural pathogensis. But each of these hypothesis cann't explain completely characters of vitiligo. Therefore some experts suggested that vitiligo is a multi-factors involved disorder. The past investigation was inclined to the autoimmune hypothesis. Recent research showed that the pigmented lesions present destruction of melanocytes and a cellar infiltrate. The most direct and convincing evidence that vitiligo is autoimmune disease is that autoantibodies to melanocyte cell surface antigens are present in the circulation of most patient with vitiligo, the level of vitiligo antibodies correlates with the extent of depigmentation and activity of the disease. Our investigative group has succeeded to identify it by immunochemical analysis and demonstrate the functional capacity to killpigment cells in vitro and do so by a mechanism-complement-dependent cytotoxicty. At present, the pigment cell antigens defined by antibodies in patients with vitiligo mainly are cellular plasm antigens, such as tyrosinase, tyrosinase related protein-1 (TRP1) and tyrosinase related protein-2 (TRP2). Other surface antigens still are unknown.Although the pathogensis of autoimmne disease don't be interpreted presently, on the basis of immunology an important link is the lost of immune tolerance to some certain autoantigens. "Toboo strains" in lymphocytes lose tabu and the B/T lymphocytes directed to specific autoantigens appear accordingly. Removing these lymphocytes is an exploration of treatment in the autoimmune disease.Our investigative group presently is cloning cDNA of the antigens related to antibodies in patients with vitiligo, and want to identify the epitopes. Then plan to make chromatography with the cloned epitope peptide to remove the correlated autoantibodies, we inject epitope peptide linked with toxic molecules to the body in order to clear the pathogenic lymphocyte strains. It maybe is succeeded to treat vitiligo.Objective To detect and analysis melanocyte surface antigens associated with vitiligo and build the foundation for purification and cloning of them. Then we will go to purify target antigen and detect the activity and construct the basis of sequencing in membrane antigen. In end, we get to clone the epitope and clear the pathogenic lymphocyte strains so that it is possible to cure vitiligo.Methods The cultered melanocytes from a health individual are used to solubilize when the cell is cultured in sixth generation. Meanwhile, usinglive cell ELIS A is to screen high titer vitiligo serum. Mixture of the pigment cell antigens and high titer vitiligo serum is western blotting, in order to detect whether there are autoantigens directed to antibodies related with vitiligo. Next step will analysis MWs of the antigens. Thus it can establish the foundation of purification. Next part will be proceeding to clone the autoantigens. Firstly, biotin-7-NHS labelled soluble melanocyte antigens. Then 50MmNH4cl must end marking. Secondly, after previous remove with Protein A-Sepharose soluble melanocyte antigens are precipitated with Protein A-Sepharose, run on SDS-10%PAGE, then blotting on the membrane. Streptavidin-POD was added to NC membrane. Lastly, the antigens are visualized by the application for the BM chemiluminescence wetern blotting Kit and exposure to films.Results 238 vitiligo sera were screened, we got 98 cases with positive result by live cell ELISA. We detected 30 vitiligo serums. In 30 individuals, there were 27 patients with vitiligo vulgaris, 26 patients being in active phase, and 3 patients with segmental vitilig...
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