炎症诱导性表达载体的表达特性

Character of inducible expression vector for excessive inflammatory response

目的 :构建一种具有自控性的炎症诱导性表达载体 ,为探索符合临床实际的抗炎基因治疗新系统奠定基础。 方法 :(1)将对炎症敏感的 SAA3启动子调控片段与报告基因 CAT片段重组 ,构建炎症诱导性表达载体 p SAA3/CAT;(2 )用脂质体转染法将表达载体 p SAA3/CAT转入体外培养细胞或动物体内 ,观察不同时间、不同剂量的炎性刺激时报告基因的表达变化 ,分析 SAA3启动子体内外转录活性和炎症诱导性。 结果 :(1)阳离子脂质体包裹表达质粒可以有效地转染肝源性细胞SMMC- 772 1和单核细胞源性细胞 U937,经活化后的 U 937细胞可迅速表达 CAT,6 h启动 ,2 4 h达峰值 ,72 h渐降至基础水平。(2 )腹腔注射脂质体包裹 DNA可有效转染外源报告基因 CAT在体内表达 ;L PS活化的基因转染小鼠肝脏中的 CAT表达与一次典型的急性反应时间一致 ,鼠肝 CAT表达水平与 L PS有显著的剂量依赖关系。 结论 :初步建立了一种具有自控性的有效抗炎基因治疗新系统。

Objective:To construct inducible expression vector for excessive inflammatory response with autocontrol, providing a new anti inflammatory genetherapy for clinical excessive inflammatory response. Methods:(1) Recombining SAA3 promoter and report gene CAT to construct inducible expression vector pSAA3/CAT. (2) Transfecting cells in vitro or animal in vivo by the mixture of pSAA3/CAT and liposome to observe expression character of report gene CAT under different inflammatory stimulators, and the transcription character of SAA3 promoter was analyzed. Results: (1) The mixture of pSAA3/CAT and liposome effectively transfected SMMC 7721 and U937 cells. Activated U937 cells expressed CAT rapidly. CAT expression was detected 6 h after LPS activation and peaked after 24 h, decreased after 72 h in U937 cells. (2) Mouse transfected with PSAA3/CAT3 lipsome mixture (ip) and LPS (ip) induced CAT reporter gene expression in liver and lung. The expression of CAT in transfected mouse was in accordance with a typical acute response, hepatic CAT expression was in proportion to the LPS doses. Conclusion:An effective strategy of anti inflammatory genetherapy with autocontrol is set up.