TGF-β1对UGBP启动子活性的影响及核心启动子区域的筛选

发布时间：2018-06-24 09:47

本文选题：肺纤维化 + UGBP　；参考：《中南大学》2012年硕士论文

【摘要】：子宫珠蛋白(uteroglobin, UG)是一种小分子分泌性蛋白,它主要由衬覆于远端细支气管的非纤毛上皮细胞--Clara细胞所分泌。UG具有抗氧化、抗炎、免疫调节、抑制成纤维细胞趋化以及抑制肿瘤细胞外基质侵袭等多种生物活性。UG的生物学作用有赖于子宫珠蛋白结合蛋白(uteroglobin binding protein, UGBP)的介导。国内外对UGBP的研究了解甚少,而有关UGBP基因转录调控的研究尚未见报道。本实验首先构建UGBP基因5’侧翼启动子区(-2234bp-+64bp)报告基因质粒,在此基础上观察TGF-β1对UGBP基因启动子活性的影响,初步筛选出UGBP核心启动子区,为进一步对UGBP转录调控原理的研究提供新的线索。方法：①采用PCR法从小鼠的总DNA中扩增出UGBP基因5’侧翼启动子区(-2234bp～+64bp),经双酶切后插入到空载体pGL3-basic中,构建报告基因重组质粒。②重组质粒转染NIH3T3和HBE细胞,转染48h后双萤光素酶检测UGBP启动子活性；③采用smad3蛋白抑制剂(SIS3)预处理,双萤光素酶检测UGBP的启动子活性。④构建不同长度UGBP启动子报告基因重组质粒,双萤光素酶检测UGBP的启动子活性。结果： 1.测序结果显示：插入到空载体pGL3-basic中的目的片段大小、方向正确,成功构建重组质粒pGL3-UGBP-FL、A、S、903、567、321。 2.双萤光素酶检测启动子活性结果显示：UGBP启动子在HBE细胞中的活性高于NIH3T3细胞中的活性(p0.05)。 3.重组质粒pGL3-UGBP-FL(1μg,2gg,4μg)转染NIH3T3和HBE细胞,2μg质粒组的UGBP启动子活性高于1μg组和4gg组(p0.05；)。 4.TGF-p1(5ng/ml)作用于HBE细胞12h、24h,双萤光素酶检测启动子活性结果显示：TGF-p1增强了UGBP的启动子活性(12h组与报告基因组比较,p0.05；24h组与报告基因组相比,p0.01；报告基因组与对照比较,p0.01)。 5.TGF-p1(10ng/ml)作用于NIH3T3细胞双萤光素酶检测启动子活性结果显示：TGF-β1增强了UGBP的启动子活性(p0.01)。 6.SIS3(3μ mol/L)预处理可以减轻TGF-β1对UGBP启动子活性的影响,SIS3+TGF-β1组与TGF-β1组比较有统计学意义(P0.05)。 7.双萤光素酶检测启动子活性结果显示：质粒pGL3-UGBP-321转染HBE细胞48h UGBP启动子活性较强,pGL3-UGBP-321质粒组与pGL3-UGBP-FL质粒组相比较没有显著差异(P0.05)；pGL3-UGBP-321质粒组与对照组相比较有统计学差异(P0.01)。结论： 1.成功构建并鉴定了一系列UGBP基因5’侧翼启动子区不同长度片段萤光素酶报告基因载体——pGL3-UGBP-FL、A、S、903bp、567bp、321bp重组质粒。 2.TGF-β1可增强NIH3T3和HBE细胞中UGBP启动子的活性。 3.TGF-β1对HBE细胞中UGBP启动子活性的影响有赖于smad细胞内信号途经的介导。 4. UGBP基因转录起始位点上游-257bp--76bp区域是UGBP基因的核心启动子区域。
[Abstract]:Uterine globin (UG) is a small molecular secretory protein. It is mainly secreted by non-ciliated epithelial cells (Clara cells) lining the distal bronchioles. UG has antioxidant, anti-inflammatory and immunomodulatory effects. Inhibition of fibroblast chemotaxis and inhibition of invasion of tumor extracellular matrix. The biological effects of UG are mediated by the uterine globin binding protein (uteroglobin binding protein,). There is little understanding of UGBP at home and abroad, but the study on transcription regulation of UGBP gene has not been reported. Firstly, the reporter gene plasmid of 5'flanking promoter region (-2234bp- 64bp) of UGBP gene was constructed, and the effect of TGF- 尾 1 on the promoter activity of UGBP gene was observed. It provides a new clue for the further study of UGBP transcriptional regulation mechanism. Methods the 5'flanking promoter region of UGBP gene (-2234bp- 64bp) was amplified from mouse total DNA by PCR method. The 5'flanking promoter region of UGBP gene (-2234bp- 64bp) was inserted into the empty vector pGL3-basic and transfected into NIH3T3 and HBE cells. After 48 hours of transfection, the recombinant plasmid of different length of UGBP promoter reporter gene was constructed by pretreatment with smad3 protein inhibitor (SIS3) and double luciferase detection of promoter activity of UGBP promoter. The promoter activity of UGBP was detected by double luciferase. Results: 1. The results of sequencing showed that the target fragment inserted into the empty vector pGL3-basic was in the right direction and the recombinant plasmid pGL3-UGBP-FLA was successfully constructed. Double luciferase assay showed that the activity of the 1: UGBP promoter in HBE cells was higher than that in NIH3T3 cells (p0.05). The activity of the recombinant plasmid pGL3-UGBP-FL (4 渭 g) transfected NIH3T3 and HBE cells with 2 渭 g plasmid was higher than that of 1 渭 g group and 4gg group (p0.05). 4. TGF-p1 (5ng/ml) acted on HBE cells for 12h and 24h. The results of double luciferase assay showed that TGF-p1 enhanced the promoter activity of HBE cells. 5. TGF-p1 (10ng/ml) acted on NIH3T3 cells to detect the promoter activity. The results showed that: TGF- 尾 1 enhanced the promoter activity of UGBP (p0.01). 6. Pretreatment with SIS3 (3 渭 mol / L) reduced TGF- 尾 1 to UGBP promoter. The activity of SIS3 TGF- 尾 1 group was significantly higher than that of TGF- 尾 1 group (P0.05). The results of double luciferase assay showed that the activity of UGBP promoter in plasmid pGL3-UGBP-321 was stronger than that in pGL3-UGBP-321 plasmid group and pGL3-UGBP-FL plasmid group (P0.05). There was no significant difference between pGL3-UGBP-321 plasmid group and control group (P0.01). Conclusion: 1. A series of different length luciferase reporter vector pGL3-UGBP-FLA-Agn-903bp517bpTGF- 尾 1 were successfully constructed and identified. 2. TGF- 尾 1 could enhance the activity of UGBP promoter in NIH3T3 and HBE cells. 3. The effect of TGF- 尾 1 on the activity of UGBP promoter in HBE cells depends on the signal pathway in smad cells. 4. The upstream of UGBP transcription initiation site-257 BP-76 BP region is the core promoter region of UGBP gene.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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