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目的 :了解在氧化低密度脂蛋白 (Ox LDL)刺激下 ,转化生长因子 β1(TGF β1)基因启动子中顺式作用元件的应答情况。研究在大鼠肾系膜细胞中 ,Ox LDL对TGF β1基因启动子转录活性的影响作用。 方法 :用聚合酶链反应 (PCR)、酶切和亚克隆的方法 ,构建出 5个TGF β1启动子缺失体—虫荧光素酶 (luciferase)报告基因 ,利用其瞬时转染系膜细胞并检测luciferase相对活性。 结果 :在Ox LDL(10 0mg/L)刺激下 ,luciferase的活性 12h即出现 ,并于 36h达到平台期。对Ox LDL刺激的应答 ,TGF β1启动子 15 5 0到 845之间可能存在着增强子 , 6 2 9到 4 2 2之间则可能有抑制子。计算机软件分析表明 ,前者内有一转录激活蛋白 1(AP 1)精确识别位点。 结论 :在大鼠肾系膜细胞TGF β1基因启动子中 ,AP 1结合序列 (TGAGTCA)可能是应答Ox LDL的顺式作用元件。Ox LDL上调TGF β1的表达至少部分是通过AP 1蛋白来介导的 ,且Ox LDL可能通过蛋白激酶C(PKC)信号通路激活AP 1从而正调控TGF β1基因的转录过程。
OBJECTIVE: To investigate the response of cis-acting elements in the transforming growth factor-β1 (TGFβ1) gene promoter stimulated by oxidized low-density lipoprotein (Ox LDL). To investigate the effect of OxLDL on the transcriptional activity of TGFβ1 promoter in rat mesangial cells. Methods: Five TGF β1 promoter deletion luciferase reporter genes were constructed by polymerase chain reaction (PCR), restriction enzyme digestion and subcloning. The luciferase reporter gene was transiently transfected into mesangial cells and detected Relative luciferase activity. Results: The luciferase activity was induced by Ox LDL (10 0 mg / L) for 12 h and reached plateau at 36 h. In response to Ox LDL stimulation, enhancers may exist between 150 and 845 of the TGFβ1 promoter, and suppressors may be present between 62 and 42.2. Computer software analysis showed that the former had a transcription activator 1 (AP1) precise recognition site. CONCLUSION: AP1 binding sequence (TGAGTCA) may be a cis-acting element in response to Ox LDL in the TGF β1 gene promoter of rat mesangial cells. Ox LDL up-regulates the expression of TGFβ1 mediated at least in part by AP 1 protein, and Ox LDL may positively activate AP 1 through the protein kinase C (PKC) signaling pathway to positively regulate the transcription of TGFβ1.