Detection Autoantibodies To Human MCHR1 In Sera Of Patients With Vitiligo And Prediction, Expression Of B Cell Epitopes Of Human MCHR1

Vitiligo is a depigmented disorder characterized by the presence of milk-whiter skin maculae, which due to lack of melanocytes in the respective areas. Recently, its incidence rate increased. Why and how melanocytes disappear to induce the characteristic achromic lesions is not fully understood. There are three major hypotheses for the pathogenesis of vitiligo. One of the most longstanding and popular hypotheses considers vitiligo as an autoimmune disease(a). The neural hypotheses suggest that accumulation of a neurochemical substance decreases melanin production(b). The biochemical hypothesis implicates autocytotoxic metabolites of melanin synthesis in the destruction of melanocytes(c). Other possible aetiological factors, such as a deficiency in unidentified melanocyte growth factors(d), an intrinsic defect of structure and function of melanocytes(e) or genetic factors(f) were proposed to be involved in the depigmentation process. The different causal factors could therefore all contribute in variable proposed in the "convergence theory". The most direct and convincing evidence for vitiligo as autoimmune disease is that about 10% patients had a family history of vitiligo(a), the depigmented lesions present destruction of melanocytes and a Cellular infiltrated (b), and autoantibodies to melanocyte cell antigens are present in the circulation of most patient with vitiligo and the level of vitiligo related antibodies correlates with the extent of depigmentation and activity of the disease(c).The outbreak of autoimmune disease is link with the lost of immunetolerance to some certain autoantigen. "Toboo strains" in lymphocytes lose tabu and the B/T lymphocytes directed to specific autoantigens appear accordingly to induce cells and organization destruction. Removing these lymphocytes is an exploration of treatment in the autoimmune disease.Our investigative group presently is cloning cDNA of the vitiligo related antigens and want to identify the epitopes. Then we plan to use chromatography with the cloned Epitopes peptide to remove the correlated autoantibodies and inject Epitopes peptide linked with toxic molecules to the body in order to clear the pathogenic lymphocyte strains. It maybe succeeded in treating vitiligo.The vitiligo-related antigen concerns antibodies to non-pigment cell antigens (common tissue antigens), cytoplasmic pigment cell antigens and pigment cell surface antigens. Antibodies to surface and cytoplasmic antigens of melanocytes have generated special interest among investigators. Tyrosinase and TRP-1 and TRP-2 are key enzymes involved in melanin synthesis primarily localized to melanosomes. Using immunoprecipitation, immunodominant antigens on the membrane of melanocyte were initially characterized with a molecular weight of 40-45, 75, 90 KD and occasionally of 35, 150KD. But the identity of these antigens remains unclear. MCHR-1 is the firstly found as vitiligo related antigen located on melanocyte membrane, which cause more attention. Because there are anti-MCHRl antibodies in sera of patients with vitiligo, MCHRl perhaps play an important role in the pathogenesis of vitiligo. It is very significant to detect autoantibodies to human melanin-concentrating hormone receptor 1 in sera of patients with vitiligo and identify B cell epitopes of human MCHRl which are recognized by vitiligo sera.The first objective of our research is to detect circulating autoantibodies to human MCHRl in sera of patients with vitiligo and evaluate its relationship to the autoantibodies to melanocytes surface antigen in the same sera. After constructing a eukaryotic expression vector pcDNA3.1/MCHRl for expression of MCHRl, it was identified by restriction endonuclease digestionand nucleotide sequencing. Then the vector pcDNA3.1/MCHRl was transfected into CHO cells by mixing with lipofectamine 2000 reagent to obtain stable expression. The expression of MCHR1 was identified by RT-PCR, ELISA and Western-blot. Then the autoantibodies to MCHR1 and melanocyte surface antigens were measured in sera of...