【作者】 曾亮；

【导师】 雷霆；

【作者基本信息】 华中科技大学 ， 外科学， 2008， 博士

【摘要】 第一部分GHS-R启动子区缺失突变体的克隆及鉴定目的构建一系列GHS-R基因5’侧翼启动子区不同长度缺失突变体的萤火虫荧光素酶报告基因增强子载体。方法以原代培养的人GH腺瘤细胞基因组DNA为模版,PCR扩增GHS-R基因5’侧翼启动子区不同长度的片段,再定向克隆到pGL3-Enhancer载体中。结果通过酶切鉴定和基因测序,证明成功地构建了pGL3-Enhancer A~F克隆重组体。结论为体外研究GHS-R基因的转录调节提供了新的手段,为下一步在垂体GH腺瘤中GHS-R基因启动子的干预研究奠定了基础。第二部分GHS-R启动子区初步功能分析的实验研究目的利用构建好的含有人GHS-R启动子区不同长度的缺失突变体的重组荧光素酶报道载体,对GHS-R启动子区的结构和功能进行初步的分析。方法①将含有人GHS-R最短启动子区片段的pGL3-Enhancer-A和最长启动子区片段的pGL3-Enhancer-F质粒DNA分别转染293细胞、95D细胞和GH3细胞。②用双荧光素酶报道系统分别检测启动子活性,判断GHS-R启动子的细胞特异性。③将含有不同长度的人GHS-R启动子区缺失突变体的荧光素酶报道载体质粒转染GH3细胞。④用双荧光素酶报道系统检测各个重组子的启动子活性,筛选GHS-R启动子区重要的转录调控区域。结果①转染pGL3-Enhancer-A质粒DNA后,各组细胞间启动子相对活性比较,统计学上无显著性差异(p>0.05);转染pGL3-Enhancer-F质粒DNA后,GH3细胞组启动子活性较293细胞组和95D细胞组有明显提高(p<0.01)。②含有不同长度的人GHS-R启动子区缺失突变体的荧光素酶报道载体质粒转染GH3细胞后启动子活性分析结果为:pGL3-Enhancer-B相对活性(8.4±0.86)与pGL3-Enhancer-A相对活性(2.3±0.21)相比,有显著升高,两组比较p<0.01;pGL3-Enhancer-C相对活性(5.2±0.30)与pGL3-Enhancer-B相对活性(8.4±0.86)相比,有明显下降(p<0.05);pGL3-Enhancer-D(10.6±0.54)相对活性与pGL3-Enhancer-C(5.2±0.30)相对活性相比,有明显升高,两组比较p<0.05;pGL3-Enhancer-E(14.1±0.74)相对活性与pGL3-Enhancer-D相对活性(10.6±0.54)相比,有明显升高,两组比较p<0.05;pGL3-Enhancer-F相对活性(12.9±0.23)与pGL3-Enhancer-E(14.1±0.74)相比,有所降低,但该变化无统计学意义(p>0.05)。结论人GHS-R启动子(-1008~+10)能在GH3细胞中特异性表达活性,而在293细胞和95D细胞中无明显作用;人GHS-R启动子区-168~+10区域之间无作用明显的启动子调控元件;在人GHS-R启动子上游-254~-168之间,-625~-355之间,-910~-625之间存在正性调控区域;而在-355~-254之间存在负性调控区域。第三部分GHS-R启动子区激素相关顺式作用元件的实验研究目的研究GHS-R启动子的激素调控,明确GHS-R启动子区激素相关顺式作用元件的基因座位。方法①用不同试剂干预有启动子活性pGL3-Enhancer-F质粒DNA瞬时转染GH3细胞。②双荧光素酶报告基因系统检测启动子活性,判定有效的干预试剂。③在有或无氢化可的松、T3和雌二醇干预下,pGL3-Enhancer-A~F载体及空载体转染GH3细胞。④用双荧光素酶报道系统检测各个重组子的启动子活性,判定启动子区激素相关顺式作用元件的特异性序列。结果①氢化可的松作用组,干预前后转pGL3-Enhancer-F载体较转pGL3-Enhancer空载体相对荧光素酶活性比值明显下降; T3作用组和雌二醇作用组,干预前后转pGL3-Enhancer-F载体较转pGL3-Enhancer空载体相对荧光素酶活性比值明显上升;余试剂作用组干预前后相对荧光素酶活性比值在统计学上无明显差异。②在有无氢化可的松作用时,pGL3-Enhancer-C组启动子相对活性比值(0.7±0.10)与pGL3-Enhancer-B组启动子相对活性比值(1.2±0.17)相比,有显著下降;在有无T3作用时,pGL3-Enhancer-B组启动子相对活性比值(1.8±0.22)与pGL3-Enhancer-A组启动子相对活性比值(1.2±0.16)相比,有明显地提高;在有无雌二醇作用时,pGL3-Enhancer-B组启动子相对活性比值(2.0±0.17)与pGL3-Enhancer-A组启动子相对活性比值(1.2±0.11)相比,有明显地提高。结论在人GHS-R启动子区(-355~-254)存在氢化可的松相关的顺式作用元件,且能被氢化可的松负性调控;在GHS-R启动子区(-254~-168)存在T3和雌二醇相关的顺式作用元件,且分别能被T3和雌二醇所正性调控。更多还原

【Abstract】 PartⅠConstructing and Identifying the GHS-R Promoter Deletion MutantsObjective: To construct a series of firefly luciferase report gene enhancer vectors for 5’flanking promoter region of GHS-R gene.Methods: A series of DNA fragments of 5’flanking promoter region of GHS-R gene were amplified from the genomic DNA of primary cultured human pituitary somatotrophinomas cells in PCR. The PCR products were then cloned into the pGL3-Enhancer vector.Results: Restriction enzymes digestion and nucleotide sequencing confirmed that the recombinant plasmids pGL3-Enhancer A~F had been constructed.Conclusion: A new methods was provided to study the transcription regulation of GHS-R gene in vitro. A foundation for further study of GHS-R gene promoter in human pituitary somatotrophinomas was established. PartⅡPrimary Function Analysis of GHS-R PromoterObjective: To analyze the structure and function of GHS-R promoter region primarily, using the constructed recombinant reporter gene promoter-pGl3-enhancer vectors containing different length of GHS-R promoter deletion mutants.Methods:①The recombinant plasmid pGL3-Enhancer-A containg the shortest length of human GHS-R promoter deletion mutants and pGL3-Enhancer-F containg the longest length of human GHS-R promoter deletion mutants were transfected into 293 cell, 95D cell and GH3 cell with the liposomes.②The promoter activities of different recombinant plasmids were detected by the Dual Luciferase Reporter Assay System, so the cell specificity of GHS-R promoter was identified.③The recombinant plasmids containg different length of human GHS-R promoter deletion mutants were transfected into GH3 cell.④The promoter activities of different recombinant plasmids were detected by the Dual Luciferase Reporter Assay System, then the important transcriptional regulation region of GHS-R promoter were identified.Results:①After transfected pGL3-Enhancer-A plasmid, we found that there was not significant difference between the promoter activities of various cell in statistics(p>0.05). After transfected pGL3-Enhancer-F plasmid, we found that the promoter activity of GH3 cell increased markedly compared with 293 cell and 95D cell.②According to the Dual Luciferase Reporter Assay System, we found that the promoter activities increased markedly between pGL3-Enhancer-B(8.4±0.86)and pGL3-Enhancer-A(2.3±0.21),p<0.01; pGL3-Enhancer-D(10.6±0.54) and pGL3-Enhancer-C(5.2±0.30), p<0.05; pGL3-Enhancer-E(14.1±0.74)and pGL3-Enhancer-D(10.6±0.54), p<0.05, and also observed that the promoter activities declined significantly between pGL3-Enhancer-C(5.2±0.30)and pGL3-Enhancer-B(8.4±0.86), p<0.05, the promoter activities declined between pGL3-Enhancer-F(12.9±0.23)and pGL3-Enhancer-E(14.1±0.74), but there was not significant difference, p>0.05.Conclusion: Human GHS-R promoter(-1008~+10)has specific expression activity in GH3 cell; There were not regulation elements between -168~+10 in the upstream of the GHS-R promoter; there was a positive transcriptional regulation region from -254 to -168, -625 to -355, -910 to -625 respectively, and a negative transcriptional regulation region between -355 to -254 in the upstream of the human GHS-R promoter. PartⅢExperimental study of hormone related cis-element in the GHS-R promoter regionObjective: To investigated the hormone regulation of GHS-R promoter, and identify the specific sequences of hormone related cis-element in the GHS-R promoter region.Methods:①Using various agents interfered the process that pGL3-Enhancer-F plasmid was transiently transfected GH3 cells.②We analyzed the effects of various agents by detecting the activity of promoter.③Plasmids containing progressively decreasing amount of GHS-R promoter region upstream of the luciferase gene were transiently transfected into GH3 cells, with or without hormonal treatment.④The promoter activities of different recombinant plasmids were detected by the Dual Luciferase Reporter Assay System, so the specific sequences of the hormone related cis-elements in the GHS-R promoter region were identified.Results:①Treatment with hydrocortisone significantly inhibited the promoter activities of pGL3-Enhancer-F; treatment with T3 orβ-estradiol significantly enhanced the pGL3-Enhancer-F; and treatment with forskolin, somatostatin, TPA and IGF-1 did not significantly influence activity of the GHS-R promoter region analyzed.②With or without hydrocortisone , we observed that the ratio of relative activity declined significantly between pGL3-Enhancer-C(0.7±0.10)and pGL3-Enhancer-B(1.2±0.17); with or without T3, we found that ratio of relative activity increased markedly between pGL3-Enhancer-B(1.8±0.22)and pGL3-Enhancer-A(1.2±0.16); with or withoutβ-estradiol, we observed that ratio of relative activity increased markedly between pGL3-Enhancer-B(2.0±0.17)and pGL3-Enhancer-A(1.2±0.11).Conclusion: A negative glucocorticoid-responsive element may be located in the GHS-R promoter region between -355 and -254; Positive thyroid and estrogen responsive elements may be located in the GHS-R promoter region between -254 and -168.更多还原