| It has been aclaimed that natural autoantibodies (NAAs), which are present in sera of healthy individuals , play an important role in maintaining homeostasis of inner envionment . Instead of "horror autoxicus, NAA is now regarded as important components of normal network of autoantibody which participate in modulation of immune homeostasis. Up to date, several hundreds kinds of NAA recognizing different self-antigens have been documented, including anti-keratin autoantibody(AK auto Ab).Anti-keratin autoantibody (AK auto Ab), circulating autoantibody against epidermal keratin, has proved to involve in physilogical and pathological process of skin. Since AK auto Ab was first found by Krogh in1970, many researchers have carried out a lot of work on its production , function and significance. For example, it was reported by Iwatsuki that AK auto Ab could beproduced spontaneously by mouse spleen cells without deliberate immunization. In vitro studies by Hinter showed that AK auto Ab could promote the phagocytosis of intermediate filament aggregates by human monocytes and polymorphonuclear neutrophils. Since 1980's, our group have been devoting to the studies of AK auto Ab. The main advances we have achieved are as follows: (1) AK auto Ab was proved to exist extensively in normal people and various animals and was related to process of physiological and pathological process of skin. (2)AK auto Ab was found to inhibit proliferation of keratinocytes (KC) and production of IL-6 and IL-8 by KC, as well as induce apoptosis of KC in vitro. (3)Four strains of hybridomas secreting AK auto Ab were obtained by fusions of B lymohocyte cells from unimmunized mouse with myloma cells, and the variable region genes were found to derive from germline gene. ã’everal strains of genetic engineering anti-keratin antibodies were obtained from phage antibody library. (5) Clinical study on AK auto Ab showed that it has promising prospect in treatment of psoriasis.Of our studies, the most important one is that AK auto Ab could inhibit proliferation of keratinocytes and squamous cells in vitro. Besides AK auto Ab from sera by affinity chromatograph, monoclonal anti-keratin antibody from mouse and engineering antibody also showed similar inhibitory pattern. The inbitory mechanism of AK auto Ab remains to be unclear. Previous studies showed AK auto Ab reacted with keratin in fixed cells(dead cells). And other studies showed that AK auto Ab does not have theinhibtory effect on cells that do not express keratin. So we deduced that binding with keratin after entering into live cells may underly the inhibitory mechanism. However, how could AK auto Ab react with keratin of living cells? And what are the sequential events after AK auto Ab's binding to keratin resulting in proliferation suppress. All these questions catch our attentions.It had been widely accepted that the binding sites of antibodies were limited to extracellar components in live cells. The intracellar components were prohibited to react with antibody. But recent advances revealed that antibodies could penetrate into the living cells and react with antigen within nucleolus and plasma, disturbing the normal functions and inducing apoptosis or death of cells. Based on the above comments, we speculate that AK auto Ab may react with keratin of living cells after entering into the cells. To confirm this assumption, we made a series of observation to learn the transmembrane penetrating activity of AK auto Ab by different methods of SABC, confocal microscope and electron microscope. We also observe the internization of genetic-engineering Fab antibody to identify if the internization depends on Fc receptor.On the base of these researches , we studied the effect of anti-keratin antibody on Tea cells, and detected the changes of phosphotyrosine of keratin and Phospho- p38 MAPK. We aimed at exploring the effects of AK auto Ab on cell and the underlying mechanism. The main results are as follows:The main results are as follows:The first part: penetrat...
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