本文選題：肺纖維化 + UGBP��； 參考：《中南大學(xué)》2012年碩士論文

【摘要】：子宮珠蛋白(uteroglobin, UG)是一種小分子分泌性蛋白,它主要由襯覆于遠(yuǎn)端細(xì)支氣管的非纖毛上皮細(xì)胞--Clara細(xì)胞所分泌。UG具有抗氧化、抗炎、免疫調(diào)節(jié)、抑制成纖維細(xì)胞趨化以及抑制腫瘤細(xì)胞外基質(zhì)侵襲等多種生物活性。UG的生物學(xué)作用有賴于子宮珠蛋白結(jié)合蛋白(uteroglobin binding protein, UGBP)的介導(dǎo)。國(guó)內(nèi)外對(duì)UGBP的研究了解甚少,而有關(guān)UGBP基因轉(zhuǎn)錄調(diào)控的研究尚未見報(bào)道。本實(shí)驗(yàn)首先構(gòu)建UGBP基因5’側(cè)翼啟動(dòng)子區(qū)(-2234bp-+64bp)報(bào)告基因質(zhì)粒,在此基礎(chǔ)上觀察TGF-β1對(duì)UGBP基因啟動(dòng)子活性的影響,初步篩選出UGBP核心啟動(dòng)子區(qū),為進(jìn)一步對(duì)UGBP轉(zhuǎn)錄調(diào)控原理的研究提供新的線索。 方法：①采用PCR法從小鼠的總DNA中擴(kuò)增出UGBP基因5’側(cè)翼啟動(dòng)子區(qū)(-2234bp～+64bp),經(jīng)雙酶切后插入到空載體pGL3-basic中,構(gòu)建報(bào)告基因重組質(zhì)粒。②重組質(zhì)粒轉(zhuǎn)染NIH3T3和HBE細(xì)胞,轉(zhuǎn)染48h后雙螢光素酶檢測(cè)UGBP啟動(dòng)子活性；③采用smad3蛋白抑制劑(SIS3)預(yù)處理,雙螢光素酶檢測(cè)UGBP的啟動(dòng)子活性。④構(gòu)建不同長(zhǎng)度UGBP啟動(dòng)子報(bào)告基因重組質(zhì)粒,雙螢光素酶檢測(cè)UGBP的啟動(dòng)子活性。 結(jié)果： 1.測(cè)序結(jié)果顯示：插入到空載體pGL3-basic中的目的片段大小、方向正確,成功構(gòu)建重組質(zhì)粒pGL3-UGBP-FL、A、S、903、567、321。 2.雙螢光素酶檢測(cè)啟動(dòng)子活性結(jié)果顯示：UGBP啟動(dòng)子在HBE細(xì)胞中的活性高于NIH3T3細(xì)胞中的活性(p0.05)。 3.重組質(zhì)粒pGL3-UGBP-FL(1μg,2gg,4μg)轉(zhuǎn)染NIH3T3和HBE細(xì)胞,2μg質(zhì)粒組的UGBP啟動(dòng)子活性高于1μg組和4gg組(p0.05；)。 4.TGF-p1(5ng/ml)作用于HBE細(xì)胞12h、24h,雙螢光素酶檢測(cè)啟動(dòng)子活性結(jié)果顯示：TGF-p1增強(qiáng)了UGBP的啟動(dòng)子活性(12h組與報(bào)告基因組比較,p0.05；24h組與報(bào)告基因組相比,p0.01；報(bào)告基因組與對(duì)照比較,p0.01)。 5.TGF-p1(10ng/ml)作用于NIH3T3細(xì)胞雙螢光素酶檢測(cè)啟動(dòng)子活性結(jié)果顯示：TGF-β1增強(qiáng)了UGBP的啟動(dòng)子活性(p0.01)。 6.SIS3(3μ mol/L)預(yù)處理可以減輕TGF-β1對(duì)UGBP啟動(dòng)子活性的影響,SIS3+TGF-β1組與TGF-β1組比較有統(tǒng)計(jì)學(xué)意義(P0.05)。 7.雙螢光素酶檢測(cè)啟動(dòng)子活性結(jié)果顯示：質(zhì)粒pGL3-UGBP-321轉(zhuǎn)染HBE細(xì)胞48h UGBP啟動(dòng)子活性較強(qiáng),pGL3-UGBP-321質(zhì)粒組與pGL3-UGBP-FL質(zhì)粒組相比較沒有顯著差異(P0.05)；pGL3-UGBP-321質(zhì)粒組與對(duì)照組相比較有統(tǒng)計(jì)學(xué)差異(P0.01)。 結(jié)論： 1.成功構(gòu)建并鑒定了一系列UGBP基因5’側(cè)翼啟動(dòng)子區(qū)不同長(zhǎng)度片段螢光素酶報(bào)告基因載體——pGL3-UGBP-FL、A、S、903bp、567bp、321bp重組質(zhì)粒。 2.TGF-β1可增強(qiáng)NIH3T3和HBE細(xì)胞中UGBP啟動(dòng)子的活性。 3.TGF-β1對(duì)HBE細(xì)胞中UGBP啟動(dòng)子活性的影響有賴于smad細(xì)胞內(nèi)信號(hào)途經(jīng)的介導(dǎo)。 4. UGBP基因轉(zhuǎn)錄起始位點(diǎn)上游-257bp--76bp區(qū)域是UGBP基因的核心啟動(dòng)子區(qū)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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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本文編號(hào)：2061108