| Psoriasis is a chronic, recurrent skin disorder, which do great harms to human physical and mental health. In China, it is estimated that the total number of patients affected by psoriasis amounts to 5, 000, 000. In recent years, the incidence of psoriasis tends to increase gradually. Due to the complexity of the pathogenesis, psoriasis is not treated effectively. Numerous investigators in the world are focusing on exploring and developing more effective, specific, new drugs with little adverse effect.Psoriasis is characterized by epidermal hyperplasia, abnormal secretion of cytokines and expression of immune phenotype. To date, several lines ofevidences have proved that anti-keratin auto antibody (AK auto Ab) can inhibit the proliferation of keratinocytes dose-dependently in vitro. In addition, AK auto Ab can also reduce the IL-6 and IL-8 expression of keratinocytes. AK auto Ab, purified from pooled sera of healthy individuals, can promote the healing of psoriasis-like lesions. When the patients of psoriasis were given immunoglobulin containing AK auto Ab intravenously, psoriatic lesions were observed to remarkably improve. Previous studies strongly indicated that AK auto Ab might be a promising biological drug in the treatment of psoriasis.When attempting to treat psoriasis by AK auto Ab, two challenges are met. Firstly, the purification of AK auto Ab from pooled sera was hindered by limited source and potential safety problems of blood sample. Secondly, the heterogeneity of monoclonal AK auto Ab generated by mouse hybridoma are also limited its clinical application. The technique of genetic engineering is a promising method to overcome the above obstacles. Our previous studies have screened a clone of human anti-keratin antibody (FK315) from a semisynthetic phage antibody library, and successfully obtained its soluble expressing Fab fragments. To explore it as a drug for treating psoriasis, the binding characteristics of FK315 and its effect on keratinocytes must be learned in detail. In present studies, the binding localization of the Fab fragment in different tissues and cells were identified. Besides, the recognized keratin bands bind by FK315 was also explored by immunoblot. Lastly, the effect of the antibody on cultured keratinocytes was also observed.Firstly, we obtained an enough amounts of FK315 by prokaryotic expression and proved that it has good specificity and high affinity to keratin.Secondly, a series of lesions (normal skin and tissues, psoriasis, squamouscell carcinoma, basal cell carcinoma, seborrheic keratosis and malignant melanoma) were collected to identify the binding characteristics of the antibody by immunohistochemistry. Our study showed that anti-keratin antibody FK315 did not bind to normal epidermis. But the incubation of anti-keratin Fab to the tissue of normal esophagus, bronchus and liver resulted in immunopositive reaction. With regard to some pathological lesions, the immunoreactivity was detected in the whole epidermis of psoriatic lesion, squamous cell carcinoma, basal cell carcinoma and seborrheic keratosis. Among the different layers, the basal cell layers of psoriatic lesions showed the strongest immunorectivity to the Fab fragment. The immunorectivity was localized mainly in the cytoplasm of squamous cell carcinoma, basal cell carcinoma and seborrheic keratosis. The Fab fragment stained negatively to lesions of malignant melanoma. In all the lesions, The Fab fragment stained positively to hair follicle.Thirdly, the corresponding keratin band recognized by FK315 was identified by immunoblot. The recognized keratin band by the antibody was similar to monoclonal antibody only against keratin 17. Analysis through gel analyzer software further proved that the recognized molecular weight by the Fab is 46 000. In addition, monoclonal anti-keratin 17 antibody significantly inhibited the binding of FK315 to keratin antigen by competition binding ELISA, which further confirmed that the Fab reacted with keratin 17.Finally, we added the antibody of dif...
|
PDF Full Text Request