衰老细胞中调控胰岛素样生长因子结合蛋白-3的表达

An expression element of a new controlling insulin growth factors binding protein-3 (IGFBP-3) in senescent cells

目的:研究在衰老细胞中调控胰岛素样生长因子结合蛋白-3(IGFBP-3)表达增加的影响因素。方法:用Northernblot的方法显示IGFBP-3基因的表达在年轻和衰老的2BS细胞中存在差异;PCR扩增出人类IGFBP-3上游包括5′-UTR区的2kb的序列并用酶切得到4组不同长短的IGFBP-3启动子片段;将各片段用Effectene Transfection Reagent试剂盒转染到年轻和衰老细胞中,测出几组构建基因片段的启动活性,确定可调控转录活性的区域;通过重叠寡核苷酸凝胶阻滞实验确定在该活性区域中的增强子元件。结果:与年轻的2BS细胞相比,衰老的2BS细胞中IGFBP-3基因的表达升高;在IG-FBP-3启动子+59到-58序列之间存在着蛋白结合位点;5′-ccagcctgccaagcagcgtgccccggttgc-3′是IGFBP-3的增强子元件。结论:在衰老的2BS细胞中IGFBP-3基因上游-37到-8的30bp序列存在一个新的增强子元件IEE(IGFBP-3enhancerelement),对IGFBP-3的表达起到调控作用。

AIM：To study the influencing factors of the increased expression of the controlling insulin growth factors binding protein-3（IGFBP-3）in the senescent cells.METHODS：Northern blot was used to show of the differential expression of the IGFBP-3 gene in the young and senescent 2BS cells;The size of 2 kb human IGFBP-3 upstream sequence including the series of the 5＇-UTR area was amplified by PCR,and four groups of IGFBP-3 promoter fragments of different lengths were obtained by enzyme digestion.The Effectene Transfection Reagent（Qiagen）kit was used to transfect the fragments into the young and senescent cells.The promoting activity of several groups of constructing gene fragments were evaluated.The area of the controlling transfection activity was determined.The enhancer element in the activity area was ascertained by superimposing the oligonucleotide gel blocking experiment.RESULTS：Compared with the young 2BS cells,the expression of the IGFBP-3 gene in the senescent 2BS cells was enhanced.There was a protein binding in the fragment site from site ＋59 to-58 of the IGFBP-3 enhancer.5＇-ccagcctgccaagcagcgtgccccggttgc-3＇ was the enhancer element of IGFBP-3.CONCLUSION：In the 30 bp fragment from site-37 to-8 of the IGFBP-3 gene upstream,there is a new IGFBP-3 enhancer element IEE,which plays a controlling role in the expression of IGFBP-3.