| 刘旭 卢晓昭 韦梦影 王姗 杨国栋.解偶联蛋白UCP1 启动子荧光素酶报告基因载体的构建[J].现代生物医学进展英文版,2015,15(25):4838-4841. |
| 解偶联蛋白UCP1 启动子荧光素酶报告基因载体的构建 |
| Construction of the Luciferase Reporter Plasmid for Uncoupling Protein 1Promoter |
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| DOI： |
| 中文关键词: 解偶联蛋白UCP1 启动子 转录调控 荧光素酶报告系统 药物筛选 |
| 英文关键词: Uncoupling protein 1 Promoter Transcriptional regulation Luciferase reporter system Drug screen |
| 基金项目:国家自然科学基金项目（31100979） |
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| 中文摘要: |
| 目的：构建解偶联蛋白UCP1 启动子荧光素酶报告基因载体，为寻找调控UCP1 表达的小分子化合物提供有效工具。方法：
从小鼠基因组DNA 中PCR扩增小鼠UCP1 启动子上游2000 bp 序列，并将该序列连接到荧光素酶报告基因载体pGL3-basic 中，
构建pGL3-UCP1 启动子。测序正确后，提取质粒，然后将上述载体与pRL-TK 载体共转染至HEK293细胞、小鼠白色脂肪前体细
胞和小鼠棕色脂肪前体细胞，48 h后裂解细胞检测荧光素酶的活性。结果：通过PCR 成功扩增获得了目的片段，并将其克隆至
pGL3-basic 中。与细胞内源UCP1 表达水平相似，荧光素酶报告系统表明构建的pGL3-UCP1 在棕色脂肪细胞中启动子活性最高，
在白色脂肪细胞中活性较低，在HEK293 细胞中基本没有活性。同时beta3 肾上腺素受体激动剂CL 316,243 同样能够上调
pGL3-UCP1 的启动子活性。结论：成功构建了小鼠UCP1 启动子荧光素酶报告基因载体，并证明在棕色脂肪细胞中，该启动子具
有很强的启动子活性，而在白色脂肪和HEK293 细胞中，启动子活性很低。该启动子报告系统有望为寻找激活UCP1 的小分子化
合物提供重要平台。 |
| 英文摘要: |
| Objective:To construct the reporter plasmid for uncoupling protein UCP1, with the purpose of screening small
molecules that can promote adipose browning.Methods:About 2000 bp fragment of mouse UCP1 promoter region was amplified using
PCR. The amplicon was further cloned into the pGL3-basic vector, which was designated as pGL3-UCP1. After confirmed by
sequencing, the correct plasmid was purified and co-transfected into HEK293, white adipocytes, and brown adipocytes. The promoter
activities in the three different cell types were compared after transfection.Results:The promoter region of UCP1 was successfully
amplified by PCR and cloned into pGL3-basic vector. Sequencing results confirmed that the inserted sequencewas correct. The
pGL3-UCP1 promoter activity in the brown adipocyte was the highest among the three cell types, while it was very low in white
adipocyte and nearly undetectable in HEK293 cells, which was consistent with the endogenous expression. In addition, beta3-adrenergic
receptor agonist CL316,243 could also increase the activity of the pGL3-UCP1 promoter reporter.Conclusion:The promoter reporter of
UCP1 has been successfully constructed. The reporter activity accurately reflect the endogenous expression level of UCP1, which has
high activity in brown adipocytes, while has low to no activity in white adipocytes and HEK293 cells. The constructed promoter reporter
can be used for future screening of small molecules that can promote browning of adipose tissue. |
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