| Natural autoantibodies can be defined as antibodies produced by healthy individual against one or more autoantigens without immunization. NAAs are the most important part of natural immunity, which maintains the homeostasis of human body , prevents autoimmunity diseases, has anti-inflammation and anti-tumor surveillance function. However, due to the limitation of related researches and research methods, little was known in the production mechanism of NAA and selection and tolerance mechanism of self-reactive B cells which produce NAAs.Development and application of antibody transgenic mice made it possible to make further study on the mechanism of tolerance of NAA. Various animal model of autoantibody transgenic mice had reveal some of the mechanism of self-tolerance and production of NAA. And for many years, our research group has been studying on so many importantfunction of anti-keratin autoantibody, which belongs to the NAA family. What's more, we fused B cells from mice which was not immuned with myeloma cells, and succeeded in screening hybridomas which can secrete anti-keratin autoantibodies. AK auto Ab from these mice is a kind of authentic NAA. Thus we constructed autoantibody transgenic mice by cloning the variable genes of AK auto Ab, which contribute to our further research on the production mechanism of NAA so greatly, we preliminary analyzed the AK auto Ab transgenic mice and found that IgM encoded by transgene are expressed in 50%~80% B cells . Level of anti-keratin IgM and total IgM in serum of transgenic mouse was analysed by ELISA, and the former was found much higher in transgenic mouse than in control mouse. And we proved that AK auto Abs are produced mainly by B cells from peritoneal cavity.However, the production mechanism of natural keratin autoreactive B cells hasn't been clarified yet. Why these B cells are not eliminated? How had they been selected? Which subset are they tended to developed into in the peripheral? What affects their development? We just did further research on the above questions by AK auto Ab transgenic mice. 1. maintenance and selection of transgenic mice Using anti-IgM/anti-IgD mAb anti-IgM/anti-IgM~a/anti-IgM~b mAbs we detected all nine lines of transgenic mice about their transgene allelic exclusion. Level of anti-keratin IgM , total IgM , IgMa and IgMb in serum of transgenic mice was analysed by ELISA.Two lines of transgenic mice with high level of allelic exclusion and increased level of anti-keratin IgM in blood serum was selected for further study.2. Analyse of the transgenic copy number and BCR densityThe number of transgenic copy was confirmed by Southern Blot. And the density of BCR was analysed by FACS using anti-IgMa antibody. Significant difference was found between two lines of Tg mice.39 mice line showed much higher level both in transgenic copy number and density of BCR in B cells than 22 mice line.3. Analyse of B cells in bone marrow from transgenic miceUsing B220 CD43 IgM AA4.1 mAbs to bind B cells, we detected B cell's surface molecular expression by FACS. The relative percentage of progenitor B cells, precursor B cells and immature B cells in the bone marrow was counted. It was founed that negative selection happened in the pre-B cells stage in transgenic mice, and the increase of density of self-reactive BCR can promote this process.4. Analyse of spleen B cells from transgenic miceUsing B220 CD21 CD23 IgM IgD mAbs to bind B cells, we detected B cell's surface molecular expression by FACS. The relative percentage of MZ-B cells, follicular B cells and immature B cells in the spleen was counted. We found that Keratin reactive B cells mainly differentiated into MZ-B cells in spleen and the increase of density of self-reactive BCR can promote this process too.5.Analyse of B cells in peritoneal cavity from transgenic miceUsing B220 CD19 CD5 mAbs and keratin labeled with fluorescence,we detected surface molecular expression of B cells from transgenic miceby FACS. The ratio of B-la cells, B-lb cells and B-2 cells in theperitoneal cavity was counted. We found that Keratin reactive B cells rich in... |
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