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缺氧诱导丝裂原因子(hypoxia-induced mitogenic factor,HIMF)是一种肺组织特异性生长因子,可刺激肺细胞增生和再生,但HIMF基因表达调控的机制尚未明确.为探索HIMF基因转录调控机制,首先以小鼠基因组DNA为模板,通过PCR扩增方法获得-348~+61bp、-302~+61bp、-131~+61bp、-68~+61bp的HIMF启动子片段,再将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子ETS-1结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染正常氧浓度条件下培养的小鼠肺上皮MLE-12和MLE-15细胞、结肠癌CT26细胞.结果发现,各HIMF启动子片段在MLE-12、MLE-15细胞中均有活性,但在CT26细胞中活性缺失;-302~-131bp区存在HIMF启动子的核心调控元件.针对该区域ETS-1转录因子结合位点进行突变或缺失,能导致HIMF启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合ETS-1.结果提示,转录因子ETS-1参与正常氧浓度下HIMF启动子活性的调控,为研究HIMF基因的转录调控机制奠定了实验基础.
Hypoxia-induced mitogenic factor (hypoxia-induced mitogenic factor, HIMF) is a lung tissue-specific growth factor that can stimulate lung cell proliferation and regeneration, but the mechanism of HIMF gene expression regulation is not yet clear.In order to explore HIMF gene transcriptional regulation Mechanism, the HIMF promoter fragment of -348 ~ + 61bp, -302 ~ + 61bp, -131 ~ + 61bp, -68 ~ + 61bp was obtained by PCR amplification using mouse genomic DNA as template, Cloned into pGL3-Basic vector to construct luciferase reporter gene vector, and prepared mutated or deleted body of transcription factor ETS-1 binding site.Related to cationic liposomes, reporter gene vectors were transiently transfected with normal oxygen MLE-12 and MLE-15 cells and colon cancer CT26 cells were cultured under the same conditions.The results showed that each HIMF promoter fragment was active in MLE-12 and MLE-15 cells, but in CT26 cells -302 ~ -131bp region exists the core regulatory element of HIMF promoter.A mutation or deletion of ETS-1 transcription factor binding site in this region can lead to a significant decrease of HIMF promoter activity; gel electrophoresis mobility test showed , This section can be combined with ETS-1. The results suggest that, The transcription factor ETS-1 participates in the regulation of HIMF promoter activity under normal oxygen concentration, which lays an experimental foundation for studying the transcriptional regulation mechanism of HIMF gene.