摘要
目的：利用构建的血清淀粉样蛋白启动子（SAA3）报告基因表达载体观察SAA3启动子／增强子应用于调控目的基因在培养细胞株的表达及对炎症刺激的响应特性，探索SAA3用于调控靶基因的可能性。方法：pSAA3CAT3采用脂质体转染U937细胞、肝细胞株SMMC7721，48h后LPS、重组rIL－6＋rIL-1、巨噬细胞活化上清（CM）攻击、测定不同时间CAT的诱导表达（ELISA法）。结果：LPS活化的转染U937细胞，6hCAT开始表达，24h达峰值，LPS活化后4．6倍增加CAT基因的表达，72h开始下降；重组rIL－6＋rIL－1联合诱导，10％、20％CM诱导转染SMMC7721细胞24h可启动报告基因表达，20％的条件活化上清诱导作用显著，6h始启动，36～48h达峰值，96h下降；本研究结果显示在0．1～62．5ng／ml的剂量范围内，rIL－6＋rIL－1联合诱导剂量依赖诱导转染U937细胞、SMMC7721细胞报告基因CAT的表达。结论：本研究证实SAA3启动子转录调控序列（－306～＋47）可启动SMMC7721、U937细胞转染报告基因CAT表达．SAA3启动子适用于构建炎症诱导性表达载体，将为基因治疗的表达调控提供新治疗策略及方法。
Objective: To study the expression characteristics of constructed pSAA3/CAT and its responsive characteristics to inflammatory stimulus in incubating cells in order to assess the possibility to modulate a target gene in gene therapy with the promotor of serum amyloid A3 gene (SAA3). Methods: U937 and SMMC7721 cell lines were transfected with lJSAA3CAT3 using Dotap liposome. The cells were incubated for 48 h and then challenged with LPS, rIL-6+IL-1 or conditioned medium (CM). The expression of CAT was detected with ELISA. Results: In U937 cells, LPS challangerapidly induced CAT expression. In the 6th hour after LPS stimulation, there was a 4. 6 fold increase of CAT expression which began to decline in the 96th hour. rIL-6+IL-1 or 20% and 10% CM were also effective to induce CAT expression in SMMC7721cells- 20% CM resulted in CAT expression in the 6th h after stimulation, reached the peak value between 36th to 48th hour and began to decrease after 96th hour. The combined stimulation of rIL-6+IL-1 (0. 1～62. 5 ng/ml) induced CAT expression in a dosage-dependent and inflammation inducible manner in U937 cells and in SMMC772l cells too. Conclusion: Our findings suggest that SAA3 is suitable to construct inflammation-inducible vector, which provides new therapeutic strategy and methods for the expression of tatget gene in gene therapy.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
1999年第10期705-708,共4页
Journal of Third Military Medical University
基金
国家自然科学基金!39600140
