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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 that regulate the expression of insulin-like growth factor binding protein-3 (IGFBP-3) in senescent cells. Methods: Northern blotting showed that the expression of IGFBP-3 gene was different in young and aged 2BS cells. The 2kb sequence upstream of human IGFBP-3 including the 5’-UTR region was amplified by PCR and was digested with four different groups Length of the IGFBP-3 promoter fragment; each fragment was transfected into young and senescent cells with Effectene Transfection Reagent kit, several groups of gene promoter activity was determined to determine the region regulating transcriptional activity; by overlapping oligonucleotides Glycine gel retardation assays determine enhancer elements in this active region. RESULTS: The IGFBP-3 gene expression was increased in senescent 2BS cells compared to young 2BS cells; there was a protein binding site between the +59 and -58 sequences in IG-FBP-3 promoter; 5’- ccagcctgccaagcagcgtgccccggttgc-3 ’is an enhancer element of IGFBP-3. CONCLUSION: There is a new enhancer element IEE (IGFBP-3 enhancer) in the 30 bp sequence from -37 to -8 upstream of the IGFBP-3 gene in aging 2BS cells, which regulates the expression of IGFBP-3.