应用细胞内PCR技术克隆白癜风自身抗体基因的研究
摘要
白癜风的病因和发病机理尚未完全阐明,有证据表明,抗黑素细胞自身抗体的产生在白癜风的发生与发展过程中起重要作用。受研究方法的限制,迄今对白癜风相关自身抗体基因的认识十分有限。噬菌体抗体库技术是抗体研究领域的重要突破,通过扩增免疫球蛋白基因并使其在噬菌体表面展示而构建抗体库,可以方便地筛选到特异性抗体。然而获得的抗体基因是体外的随机组合,并不是体内原本的轻重链搭配,在自身抗体基因的研究上存在较大的局限性。细胞内PCR(In-Cell PCR)技术可获得亲本配对的抗原结合单位,从而得到编码抗体基因的准确信息,对于自身免疫性抗体的研究具有极广阔的前景。
在本课题中,我们培养了大量正常人的黑素细胞,经相差显微镜观察细胞形态,多巴染色鉴定,保证较高的黑素细胞纯度,将其作为抗原,以活细胞ELISA法和Westonblot法对50余名白癜风患者的血
Although the pathogenesis of vitiligo has been not clarified absolutely, some evidences showed that the autoantibodies reacted with melanocyte surface antigens played a very important role during the occurrence and development of vitiligo. While have been confined by research methods, there are very less information about the gene of autoantibodies correlated with vitiligo. It is the phage-displayed Ab library that lead to a important breakthrough of the antibody research.By using the large number of applications of combinatorial library methods to the study of human disease-associated Ab ,the investigators find the high affinity and the epitope specificity of Abs.The construction of a large library of single chain Fv antibody framents involves a random assortment of heavy and light chains.Although useful for the profuction of recombinant antibodies,this
method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmine diseases.The maior question is that the Vh and Vl chain pairing of these antibodies from random combinatorial library redlect the in vivo pairing . It is in-cell PCR that help us to get the natural Vh/Vl pairing of antibodies .This in cell amplification and association produre is a potentially useful tool for the study of autoantibody gene families and Vh/Vl pairing that occurs during the autoimmine process.In the study, we cultered melanocytes from a health individual are used to solubilize when the cell is cultured in forth generation. Meanwhile, using live cell ELISA 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. About 50 vitiligo sera were screened, we got 7 cases with positive result by live cell ELISA. We detected 7 vitiligo serums. All these patients with vitiligo vulgaris,were being in active phase. We found that all 7 patients had positive strips by immunoblotting, comparatively only one health case had positive result in 10 control sera. Our methods are based on amplification and recombination of the VH and Vl genes within CD19+B cells isolated from peripheral blood lymphocytes of seven vitiligo patients who have the strong positive sera screened. The cells were fixed in 10% buffered formaldehyde and permeabilised by Proteinase K,the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction(PCR). The Ig Vh and Vl DNA assembled within the same cell using the Cre/loxP system. Nested primers were designed to amplify the known major human Vh and Vl gene familes. We obtained a
在本课题中,我们培养了大量正常人的黑素细胞,经相差显微镜观察细胞形态,多巴染色鉴定,保证较高的黑素细胞纯度,将其作为抗原,以活细胞ELISA法和Westonblot法对50余名白癜风患者的血
Although the pathogenesis of vitiligo has been not clarified absolutely, some evidences showed that the autoantibodies reacted with melanocyte surface antigens played a very important role during the occurrence and development of vitiligo. While have been confined by research methods, there are very less information about the gene of autoantibodies correlated with vitiligo. It is the phage-displayed Ab library that lead to a important breakthrough of the antibody research.By using the large number of applications of combinatorial library methods to the study of human disease-associated Ab ,the investigators find the high affinity and the epitope specificity of Abs.The construction of a large library of single chain Fv antibody framents involves a random assortment of heavy and light chains.Although useful for the profuction of recombinant antibodies,this
method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmine diseases.The maior question is that the Vh and Vl chain pairing of these antibodies from random combinatorial library redlect the in vivo pairing . It is in-cell PCR that help us to get the natural Vh/Vl pairing of antibodies .This in cell amplification and association produre is a potentially useful tool for the study of autoantibody gene families and Vh/Vl pairing that occurs during the autoimmine process.In the study, we cultered melanocytes from a health individual are used to solubilize when the cell is cultured in forth generation. Meanwhile, using live cell ELISA 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. About 50 vitiligo sera were screened, we got 7 cases with positive result by live cell ELISA. We detected 7 vitiligo serums. All these patients with vitiligo vulgaris,were being in active phase. We found that all 7 patients had positive strips by immunoblotting, comparatively only one health case had positive result in 10 control sera. Our methods are based on amplification and recombination of the VH and Vl genes within CD19+B cells isolated from peripheral blood lymphocytes of seven vitiligo patients who have the strong positive sera screened. The cells were fixed in 10% buffered formaldehyde and permeabilised by Proteinase K,the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction(PCR). The Ig Vh and Vl DNA assembled within the same cell using the Cre/loxP system. Nested primers were designed to amplify the known major human Vh and Vl gene familes. We obtained a
引文
[1] 李志强,高天文,赵小东,马翠玲 白癜风患者血清中抗黑素细胞膜表面抗原自身抗体的检测,中华皮肤科杂志,2001,34(3):268-270.
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[4] Chapal N, Bouanani M, Embleton MJ, Navarro-Teulon I, Biard-Piechaczyk M, Pau B, Peraldi-Roux S. In-cell assembly of scFv from human thyroid-infiltrating B cells. Biotechniques, 1997, 23(3): 518-524
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[2] Schmitz U, Versmold A, Kaufmann P, Frank HG.. Phage display: a molecμLar tool for the generation of antibodies—a review. Placenta, 2000, 21 (Suppl A): S106-S112
[3] Embleton MJ, Gorochov G, Jones PT, Winter G. In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobμLin heavy and light chain V-genes within single cells. Nucleic Acids Res, 1992, 20(15): 3831-3837
[4] Chapal N, Bouanani M, Embleton MJ, Navarro-Teulon I, Biard-Piechaczyk M, Pau B, Peraldi-Roux S. In-cell assembly of scFv from human thyroid-infiltrating B cells. Biotechniques, 1997, 23(3): 518-524
[5] Chapal N, Peraldi-Roux S, Bresson D, Pugniere M, Mani JC, Granier C, Baldet L, Guerrier B, Pau B, Bouanani M. Human anti-thyroid peroxidasesingle-chain fragment variable of Ig isolated from a combinatorial library assembled in-cell: insights into the in vivo situation. J Immunol, 2000, 164(8): 4162-41629
[6] Garsaud P, Boisseau-Garsaud AM, Ossondo M, Azaloux H, Escarmant P, Mab GL, Zimmermann S, Saint-Cyr A, Quist D, Helenon R, Jouannelle A. Epidemiology of vitiligo in the French West Indies (Isle of Martinique).Int J Dermatol, 2000, 39(1): 18-20
[7] Kemp EH, Waterman EA, Weetman AP. Autoimmune aspects of vitiligo. Autoimmunity, 2001, 34(1): 65-77
[8] Ortonne JP, Bose SK. Vitiligo: where do we stand? Pigment Cell Res, 1993, 6(2): 61-72
[9] Yu HS. Melanocyte destruction and repigmentation in vifiligo: a model for nerve cell damage and regrowth. J Biomed Sci, 2002, 9(6 Pt 2): 564-573
[10] Yang F, Sarangarajan R, Le Poole IC, Medrano EE, Boissy RE. The cytotoxicity and apoptosis induced by 4-tertiary butylphenol in human melanocytes are independent of tyrosinase activity. J Invest Dermatol, 2000, 114(1): 157-164
[11] Spritz R, Fain P, Bennett D. The Epidemiology and Genetics of Generalized Vitiligo.Pigment Cell Res, 2003, 16(5): 589-90
[12] Kemp EH, Waterman EA, Weetman AP. Immunological pathomechanisms in vitiligo. Expert Rev Mol Med, 2001 23(1): 1-22
[13] Handa S, Dogra S. Epidemiology of childhood vitiligo: a study of 625 patients from north India.Pediatr Dermatol, 2003, 20(3): 207-210
[14] Le Poole IC, van den Wijngaard RM, Westerhof W, Das PK. Presence or absence of melanocytes in vitiligo lesions: an immunohistochemical investigation. J Invest Dermatol, 1993 100(6): 816-822
[15] 李强 高天文 李春英 卢涛 王秋枫 白癜风相关黑素细胞膜抗原的检测及分析.中华皮肤科杂志,2003,36(1):76-78
[16] Gilhar A, Zelickson B, MLman Y, et al. In vivo destruction of melanocytes by the IgG fraction of serum from patients with vitiligo. J Invest Dermatol, 1995, 105(5): 683-686
[17] Kim SM, Lee HS, Harm SK. The efficacy of low-dose oral corticosteroids in the treatment of vitiligo patients. Int J Dermatol. 1999, 38(7): 546-550
[18] Lengagne R, Le Gal FA, Garcette M, Fiette L, Ave P, Kato M, Briand JP, Massot C, Nakashima I, Renia L, Guillet JG, Prevost-Blondel A. Pontaneous vitiligo in an animal model for human melanoma: role of tumor-specific CD8+T cells. Cancer Res, 2004,64(4): 1496-501
[19] Bystryn JC. Serum antibodies in vitiligo patients. Clin Dermatol. 1989, 7(2): 136-145
[20] Naughton GK, Eisinger M, Bystryn JC. Detection of antibodies to melanocytes in vitiligo by specific immunoprecipitation. J Invest Dermatol,1983, 81(6): 540-542
[21] Yu HS, Kao CH, Yu CL. Coexistence and relationship of antikeratinocyte and antimelanocyte antibodies in patients with non-segmental-type vitiligo. J Invest Dermatol, 1993, 100(6): .823-828
[22] Hann SK, Shin HK, Park SH, Reynolds SR, Bystryn JC. Detection of antibodies to melanocytes in vitiligo by western immunoblotting.Yonsei Med J, 1996, 37(6): 365-370
[23] Fishman P, Azizi E, Shoenfeld Y, Sredni B, Yecheskel G, Ferrone S, Zigelman R, Chaitchik S, Floro S, Djaldetti M. Vitiligo autoantibodies are effective against melanoma. Cancer, 1993, 72(8):2365-2369
[24] Norris DA, Capin L, Weston WL. The effect of epicutaneous glucocorticosteroids on human monocyte and neutrophil migration in vivo. J Invest Dermatol, 1982, 78(5):386-390
[25] Wittbjer A, Odh G, Rosengren AM, Rosengren E, Rorsman H. Isolation of soluble tyrosinase from human melanoma cells. Acta Derm Venereol, 1990, 70(4): 291-294
[26] al Badri AM, Foulis AK, Todd PM, Gariouch JJ, Gudgeon JE, Stewart DG, Grade JA, Goudie RB. Abnormal expression of MHC class II and ICAM-1 by melanocytes in vitiligo. J pathol, 1993, 169(2): 203-206
[27] Song YH, Connor E, Li Y, Zorovich B, Balducci P, Maclaren N. The role of tyrosinase in autoimmune vitiligo. Lancet, 1994, 344(8929): 1049-1052
[28] Fishman P. Tyrosinase autoantibodies: the autoantibodies.1st ed, USA, 1996: 842-845
[29] Cui J, Arita Y, Bystryn JC. Characterization of vitiligo antigens. Pig Cell Res, 1995, 8(1): 53-59
[30] Bouchard B, Del Marmol V, Jackson IJ, Cherif D, Dubertret L. MolecμLar characterization of a human tyrosinase -related - protein-2 cDNA. patterns of expression in melanocytic cells. Eur J Biochem, 1994, 219(1-2): 127-134
[31] Okamoto T, Irie RF, Fujii S, Huang SK, Nizze AJ, Morton DL, Hoon DS. Anti- tyrosinase -related protein-2 immune response in vitiligo patients and melanoma patients receiving active-specific immunotherapy. J Invest Dermatol, 1998, 111(6): 1034-1039
[32] Kemp EH, Gawkrodger DJ, Watson PF, Weetman AP.Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo sera: evidence for cross-reactive autoantibodies to tyrosinase and tyrosinase-related protein-2 (TRP-2). Clin Exp Immunol, 1997, 109(3):495-500
[33] Cui J, Harning R, Henn M, Bystryn JC. Identification of pigment cell antigens defined by vitiligo antibodies. J Invest Dermatol, 1992, 98(2): 162-165
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