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目的:探讨多形性腺瘤样基因2(PLAGL2)和甲状腺转录因子-1(TTF-1)对表面活性蛋白C(SP-C)基因启动子的共同调控作用。方法:应用定点诱变技术和体外细胞共转染后荧光素酶报告基因活性值检测技术研究PLAGL2、TTF-1对SP-C基因启动子表达活性的影响及相互作用;应用实时荧光PCR技术检测PLAGL2、TTF-1及SP-C在人胚肺和成熟小鼠肺Ⅱ型上皮细胞中的表达。结果:荧光素酶报告基因活性值检测结果显示TTF-1、PLAGL2共转染后所测SP-C基因启动子荧光素酶活性值明显低于PLAGL2共转染后SP-C基因启动子的活性值(P<0.01);SP-C基因启动子野生型质粒转染后的荧光素酶活性值明显低于其突变体质粒转染后的活性值(P<0.005);实时荧光PCR检测结果显示,PLAGL2基因的表达水平随胚肺发育成熟而逐渐减少,TTF-1和SP-C基因的表达水平则逐渐增高。结论:在肺Ⅱ型细胞中,PLAGL2和TTF-1对SP-C基因共同调控的过程中体现出一种动态平衡、相互抑制的关系。
Objective: To investigate the common regulation of surfactant protein C (SP-C) promoter by pleomorphic adeno-associated gene 2 (PLAGL2) and thyroid transcription factor-1 (TTF-1). Methods: The effects of PLAGL2 and TTF-1 on the activity of SP-C gene promoter were studied by using site-directed mutagenesis and luciferase reporter assay in vitro. Real-time PCR was used to detect the effect of PLAGL2 and TTF- Expression of PLAGL2, TTF-1 and SP-C in Lung Type Ⅱ Epithelial Cells of Human Embryonic Lung and Mature Mice. Results: The results of luciferase reporter assay showed that the luciferase activity of SP-C gene promoter after TTF-1 and PLAGL2 cotransfection was significantly lower than that of SP-C promoter after PLAGL2 cotransfection (P <0.01). The luciferase activity of wild-type plasmids transfected with SP-C gene was significantly lower than that of mutant plasmids (P <0.005). The results of real-time fluorescence PCR , The expression level of PLAGL2 gene gradually decreased with the maturation of embryo lung and the expression level of TTF-1 and SP-C gene gradually increased. CONCLUSION: In the lung type II cells, PLAGL2 and TTF-1 exert a dynamic balance and mutual inhibitory relationship during the co-regulation of SP-C gene.