摘要目的 探讨高迁移率族蛋白(HMGB1)基因第4内含子区(1144 bp~1296 bp)153 bp长度片段可能的功能以及1176 G-C突变对其功能的影响.方法 构建HMGB1基因(1144 bp~1296 bp)第4内含子区1176GG/1176CC的153 bp目的片段与荧光素酶报告基因载体重组质粒,应用脂质体介导的转染技术,将重组质粒与pRL-TK内参照质粒共同转染Hepa G2细胞,瞬时表达,利用双荧光素酶测试系统检测荧光素酶表达活性.结果 在HepG2细胞中,pGL3-Basic-C和pGL3-Basic-G重组质粒与pGL3-Basic荧光素酶活性无明显差异;pGL3-Promoter-G和pGL3-Promoter-C重组质粒与pGL3-Promoter荧光素酶活性相比P<0.01;pGL3-Promoter-G和pGL3-Promoter-C重组质粒荧光素酶活性相比P<0.01.结论 HMGB1基因内含子区(1144~1296 bp)153 bp片段具有增强子功能以及单核苷酸1176 G-C的改变可使增强子功能大大提高.

abstractsObjective To investigate the potential function of the 153 bp fragment in the intron4 (1144 bp-1296 bp)of the high-mobility group box 1 protein(HMGB1)gene and the effect of 1176 G-C mutation on its function. Methods A 153 bp target fragment of 1176GG/1176CC in the intron4 of the HMGB1 gene(1144 bp-1296 bp)and the recombinant plasmids of the luciferase reporter gene vector were constructed. The lipofectamine-mediated transfection technology was used. The Hepa G2 cells were transfected with the recombinant plasmids and pRL-TK internal reference plasmids with transient expression. The luciferase expression activity was measured using a dual luciferase assay system. Results In HepG2 cells,there were no significantly difference in the luciferase activity between the pGL3-Basic-C and pGL3-Basic-G recombinant plasmids and the pGL3-Basic. The luciferase activity of the pGL3-Promoter-G and pGL3-Promoter-C recombinant plasmids was compared with that of the pGL3-Promoter,and P<0.01. The luciferase activity of pGL3-Promoter-G was compared with that of the pGL3-Promoter-C recombinant plasmids,and P<0.01. Conclusion The 153 bp fragment of the HMGB1 intron(1144 bp-1296 bp)has an enhancer function and the changes of single nucleotide 1176 G-C could greatly improve the enhancer function.