不同活性的survivin基因启动子片段的克隆及鉴定

Cloning and identification of survivin gene promoter segments with different activities

目的检测survivin在成体干细胞等细胞中的表达差异,克隆并比较3种不同片段长度survivin启动子的活性。方法取成纤维母细胞10T1/2、成肌母细胞C2C12、真皮多能干细胞DMSC、人骨髓间充质干细胞BMSC、结肠腺癌细胞HT29、人胚肾母细胞293FT进行培养。用RT-PCR和免疫细胞化学染色检测survivin在成体干细胞和肿瘤细胞中的表达;用基因组DNA PCR和分子克隆等方法,构建不同长度的survivin基因启动子驱动荧光素酶报告基因表达的载体,经脂质体转染293FT细胞,测定相对荧光素酶活性以反映启动子活性。结果 survivin基因在正常成体干细胞中低表达,在成肌母细胞中中度表达,在结肠腺癌细胞HT29中高表达。克隆了不同片段长度的survivin基因启动子,并连接到荧光素酶cDNA上游。在293FT细胞中,987 bp和160 bp的survivin基因启动子活性高于270 bp的survivin启动子的活性。结论 survivin基因启动子由于肿瘤特异性和泛肿瘤表达的特点,可通过载体构建应用于监控和治疗移植后干细胞的恶性转化。用高活性survivin基因启动子构建的载体可能会提高其在监控和治疗中的效果。

Objective To study the expression of survivin gene in adult stem cells and the activities of 3 cloned survivin gene promoter segments with different lengths.Methods Fibroblasts（10T1/2）,myoblasts（C2C12）,dermal multifunctional cells（DMSC）,human bone mesenchymal cells（BMSC）,colonic adenoma cells（HT29）,and human nephroblasts（293FT） were cultured.Expressions of survivin gene in adult stem cells and tumor cells were detected by RT-PCR with immunochemical staining.Survivin gene promoter vector with different lengths was constructed with luciferase and transfected into 293FT with lipid some.Activity of luciferase reflecting the activity of promotors was assayed.Results The survivin gene was mildly expressed in normal adult stem cells,moderately expressed in fibroblasts,and well expressed in colonic adenoma cells（HT29）.Survivin gene promoters with different lengths were cloned and linked to the upstream of luciferase cDNA.The activity of 987 bp-and 160 bp-long survivin gene promoters was higher than that of 270 bp-long survivin gene promoter in 293FT.Conclusion The survivin gene promoter can be used in monitoring and treatment of the malignant transformation of implanted stem cells by constructing its expression vector because of the tumor-specific and general expression characteristics.The vector constructed with highly active survivin gene promoter can improve the efficacy of gene therapy.