高迁移率蛋白B1在急性肝功能衰竭中的作用及机制研究-内科学(传染病)专业论文.docxVIP

华中科技大学博士学位论文 华 中 科 技 大 学 博 士 学 位 论 文 PAGE 10 PAGE 10 对照组降低且表达时间延后；实验组 TNF-α 于 4h 开始上升， 12h 达到峰值 （334.12±39.13）pg/ml，对照组于 8h 达到峰值（473.42±22.99）pg/ml，两组比较， t=-11.387, P0.05；实验组 IL-1β 自 2h 起随时间延长逐渐增加，在 6h 达到峰值 （341.31±25.01）pg/ml，对照组于 2h 达到峰值（724.49±34.24）pg/ml，两组比较，t=-7.999, P0.05。 结论：重组 HMGB1-Abox 蛋白在体外可以降低小鼠单核细胞在受到 LPS 刺激时 HMGB1 的表达和分泌；在体内可以显著减少急性肝衰竭时肝组织 HMGB1 的表达和 分泌，有效的阻断 HMGB1 对其他早期炎性因子的促进作用。 关键词：重组 HMGB1-Abox 蛋白； 小鼠肝功能衰竭；鼠单核细胞系 RAW264. 7； TNF-α；IL-1β ABSTRACT Part Ⅰ Construction, expression and purfication of prokaryotic expression plasmids of total length HMGB1 and HMGB1-Abox Objective: To construct the total length high mobility group box 1 and high mobility group box 1-A box expression vectors, then got the his-tagged recombinant mouse HMGB1(rHMGB1) and the his-tagged recombinant mouse HMGB1-Abox (rHMGB1-Abox). Methods: Total RNA was extracted from liver tissue of mice, as the template for a polymerase chain reaction (PCR). Two primers were synthesized to amplify the entire coding sequence and to inset a BamH I site and a Xol I (Hand Ⅲ)site. PCR products were ligated into a pMD18-T vector and confirmed by restriction digestion and sequencing. Using DNA Ligation kit, the BamH I ^Xol I(Hand Ⅲ) fragment of the pMD-18T-HMGB1 was inserted into the BamH I and Xol I(Hand Ⅲ) sites of the pET-28a vector (containing a his6 tag), then got the resulting vectors, pET-28a- HMGB1 and pET-28a- HMGB1-Abox. Protein expressions were induced by adding isopropyl-1-thio-β-D-galactopyranoside (IPTG). Purification of two proteins was achieved by affinity chromatography using a Ni-NTA column and elution with buffer containing 200mM (50 mM) imidazole. Results: The pET-28a- HMGB1 and the vector pET-28a- HMGB1-Abox were confirmed by restriction digestion and sequencing. The molecular mass of purified HMGB1 and HMGB1-Abox determined by SDSwere expected based on the theoretical value of his-tagged rHMGB1 and rHMGB1-Abox. Conclusion: The prokaryotic expression plasmids of total l