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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 detect the expression of survivin in adult stem cells and other cells, and to clone and compare the survivin promoter activity of three different fragment lengths. Methods Fetal fibroblasts 10T1 / 2, myoblast C2C12, dermal pluripotent stem cells DMSC, human bone marrow mesenchymal stem cells BMSC, colon adenocarcinoma HT29 and human embryonic kidney 293FL cells were cultured. The expression of survivin in adult stem cells and tumor cells was detected by RT-PCR and immunocytochemical staining. The vectors with different lengths of survivin promoter were used to drive the expression of luciferase reporter gene by genomic DNA PCR and molecular cloning. Lipofectamine 293FT cells were transfected and the relative luciferase activity was measured to reflect promoter activity. Results The survivin gene was overexpressed in normal adult stem cells and moderate in myoblasts and was highly expressed in colon adenocarcinoma cells HT29. The survivin gene promoter of different fragment length was cloned and ligated to the upstream of luciferase cDNA. In 293FT cells, 987 bp and 160 bp survivin gene promoter activity than 270 bp survivin promoter activity. Conclusion The survivin gene promoter can be used to monitor and treat the malignant transformation of stem cells after transplantation due to the characteristics of tumor-specific and pan-tumor expression. Vectors constructed with highly active survivin gene promoters may improve their effectiveness in monitoring and treatment.