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Evaluation of alteration in DNA methylation associated with 2,3,7,8-tetrachlorodibenzo-p-diozxin (TCDD)-induced inhibition of differentiation in lipopolysaccharide (LPS)-stimulated murine splenocytes
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Evaluation of alteration in DNA methylation associated with 2,3,7,8-tetrachlorodibenzo-p-diozxin (TCDD)-induced inhibition of differentiation in lipopolysaccharide (LPS)-stimulated murine splenocytes
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Evaluation of alteration in DNA methylation associated with 2,3,7,8-tetrachlorodibenzo-p-diozxin (TCDD)-induced inhibition of differentiation in lipopolysaccharide (LPS)-stimulated murine splenocytes
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Dissertation
Evaluation of alteration in DNA methylation associated with 2,3,7,8-tetrachlorodibenzo-p-diozxin (TCDD)-induced inhibition of differentiation in lipopolysaccharide (LPS)-stimulated murine splenocytes
2010
Overview
Splenic B-cells isolated from mice injected with LPS, differentiate into antibody-producing plasma cells in vitro. Pretreatment with TCDD, a potent immunosuppressive agent, impairs B-cell differentiation. Altered DNA methylation, an epigenetic event, may play a key role in this impairment. DNA was isolated from splenocytes prepared 6 days post experiment initiation from 5–6wk old female C57BL/6 mice dosed with: TCDD, 3 or 30μg/kg, on day 0; or LPS, 25μg/mouse, on day 4; or sequentially with TCDD and then LPS on days 0 and 4, respectively. To discern regions of altered DNA methylation (RAMs), DNA was restricted with HpaII (a methylation sensitive enzyme), followed by arbitrarily primed PCR and capillary electrophoresis. The mRNA expression of selected genes (annotated RAMs or genes closely affected by them) that might affect B-cell differentiation were analyzed using qRT-PCR. LPS, 3, or 30μg/kg TCDD alone resulted in 40, 43 and 42 RAMs, respectively, while LPS challenge subsequent to 3 or 30μg/kg TCDD resulted in 34 and 39 RAMs, respectively. Interestingly, the combined treatments lead to many RAMs observed in single treatments but also many unique RAMs. Three patterns of mRNA expression were observed: no change, similar changes in all groups, and different changes based upon treatment. Collectively, this research suggests a novel epigenetic mechanism potentially regulating gene expression stimulated by LPS and TCDD exposure and important in the differentiation of B-cells in the splenocyte population.
Publisher
ProQuest Dissertations & Theses
Subject
ISBN
1124333800, 9781124333809
