發(fā)布時(shí)間：2018-04-28 09:17

  本文選題：乙型肝炎病毒 + autotaxin��； 參考：《桂林醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:乙型肝炎病毒(HBV)感染仍是全球性的主要健康問(wèn)題之一。乙型肝炎及其并發(fā)癥的發(fā)病機(jī)制需要進(jìn)一步研究。本研究探討HBV反式調(diào)節(jié)因子X(jué)蛋白(HBx)、前S2蛋白(pre-S2)對(duì)autotaxin(ATX)表達(dá)的影響及其意義,為闡明乙型肝炎及其并發(fā)癥的發(fā)病機(jī)制提供實(shí)驗(yàn)依據(jù)。方法:PCR擴(kuò)增ATX啟動(dòng)子序列和HBx基因、pre-S2基因,并分別克隆入pGL3-Enhancer和pcDNA3.1(+)中,構(gòu)建ATX啟動(dòng)子重組熒光素酶報(bào)告基因載體p GL3-ATX和HBx、pre-S2重組真核表達(dá)載體pcDNA3.1(+)-HBx、pcDNA3.1(+)-pre-S2,將重組載體共轉(zhuǎn)染HepG2細(xì)胞,通過(guò)熒光素酶活性檢測(cè)HBx、pre-S2對(duì)ATX基因啟動(dòng)子的作用。構(gòu)建穩(wěn)定表達(dá)HBx的HepG2.HBx細(xì)胞及穩(wěn)定表達(dá)pre-S2的HepG2.pre-S2細(xì)胞,Western blotting檢測(cè)ATX蛋白的表達(dá)。結(jié)果:雙酶切檢測(cè)和DNA測(cè)序均證實(shí)重組載體pGL3-ATX和pcDNA3.1(+)-HBx的構(gòu)建與設(shè)計(jì)相符。熒光素酶活性檢測(cè)顯示pGL3-ATX熒光素酶活性是空載體pGL3-basic的10倍(P0.001)。共轉(zhuǎn)染結(jié)果顯示,pcDNA3.1(+)-HBx+pGL3-ATX組、pcDNA3.1(+)-pre-S2+pGL3-ATX組的熒光素酶活性分別是空載體pcDNA3.1(+)+pGL3-ATX組的1.47倍(P0.001)和1.15倍(p0.001)。瓊脂糖凝膠電泳檢測(cè)結(jié)果顯示HBx和pre-S2基因片段分別在HepG2細(xì)胞中穩(wěn)定表達(dá)。Western blotting檢測(cè)結(jié)果顯示,HBV穩(wěn)轉(zhuǎn)細(xì)胞株HepG2.2.15細(xì)胞、穩(wěn)定表達(dá)HBx的HepG2.HBx細(xì)胞和穩(wěn)定表達(dá)pre-S2的HepG2.pre-S2細(xì)胞相較于普通HepG2細(xì)胞,其ATX蛋白的表達(dá)均增強(qiáng)。結(jié)論:1.克隆了HBx基因和ATX啟動(dòng)子序列,且ATX啟動(dòng)子序列具有啟動(dòng)子活性;2.構(gòu)建了HBx重組真核表達(dá)載體pcDNA3.1(+)-HBx和ATX啟動(dòng)子重組熒光素酶報(bào)告基因載體pGL3-ATX;3.建立了分別穩(wěn)定表達(dá)HBx和pre-S2基因片段的細(xì)胞HepG2.HBx和HepG2.pre-S2;4.HBx和pre-S2均可增強(qiáng)ATX啟動(dòng)子熒光素酶報(bào)告基因活性;5.ATX蛋白在普通肝癌HepG2細(xì)胞、穩(wěn)定表達(dá)pre-S2基因片段的HepG2.pre-S2細(xì)胞、穩(wěn)定表達(dá)HBx基因片段的HepG2.HBx細(xì)胞和HBV穩(wěn)定表達(dá)株HepG2.2.15細(xì)胞中的相對(duì)表達(dá)量依次增強(qiáng);6.HBx和pre-S2可激活A(yù)TX基因啟動(dòng)子,上調(diào)ATX的表達(dá),提示HBV感染可通過(guò)其反式調(diào)節(jié)蛋白HBx、pre-S2上調(diào)ATX表達(dá),增強(qiáng)ATX/溶血磷脂酸信號(hào)轉(zhuǎn)導(dǎo),進(jìn)而影響宿主細(xì)胞生理和病理過(guò)程。
[Abstract]:Objective: hepatitis B virus (HBV) infection is still one of the major global health problems. The pathogenesis of hepatitis B and its complications needs further study. The purpose of this study was to investigate the effect of HBV transregulatory factor X protein pre-S2 (pre-S2) on the expression of autotaxin (ATX) and to provide experimental evidence for elucidating the pathogenesis of hepatitis B and its complications. Methods ATX promoter sequence and HBx gene pre-S2 gene were amplified by 1: PCR and cloned into pGL3-Enhancer and pcDNA3.1 (respectively). The recombinant luciferase reporter gene vectors p GL3-ATX and HBX pre-S2 were constructed, and the recombinant eukaryotic expression vector pcDNA3.1 (-pre-S2) was cotransfected into HepG2 cells. Luciferase activity was used to detect the effect of HBX pre-S2 on ATX gene promoter. HepG2.HBx cells expressing HBx stably and HepG2.pre-S2 cells expressing pre-S2 stably were constructed to detect the expression of ATX protein by Western blotting. Results: the construction of recombinant vector pGL3-ATX and pcDNA3.1 (-HBx) was confirmed by double enzyme digestion and DNA sequencing. The luciferase activity of pGL3-ATX was 10 times that of pGL3-basic. The results of co-transfection showed that the luciferase activity of pcDNA3.1 (pre-S2 pGL3-ATX group) was 1.47 times as much as that of empty vector pcDNA3.1 (P0.001) and 1.15 times of p0.001 of pGL3-ATX group. The results of agarose gel electrophoresis showed that HBx and pre-S2 gene fragments were stably expressed in HepG2 cells. Western blotting analysis showed that HepG2.2.15 cells, HepG2.HBx cells expressing HBx stably and HepG2.pre-S2 cells expressing pre-S2 stably were compared with HepG2 cells. The expression of ATX protein was enhanced. Conclusion 1. HBx gene and ATX promoter sequence were cloned, and ATX promoter sequence had promoter activity. The recombinant eukaryotic expression vector pcDNA3.1 (ATX promoter pGL3-ATXF3) was constructed, and the recombinant luciferase reporter gene vector pGL3-ATX3 was constructed. HepG2.HBx and HepG2.pre-S2O4.HBX and pre-S2 could enhance the activity of luciferase reporter gene of ATX promoter in HepG2 cells, and HepG2.pre-S2 cells expressing pre-S2 gene fragments stably. The relative expression of HBx gene fragments in HepG2.HBx cells and HepG2.2.15 cells in HBV stable expression strain was enhanced in turn. HBX and pre-S2 could activate ATX gene promoter and up-regulate ATX expression, suggesting that HBV infection could up-regulate ATX expression through its transregulatory protein HBxS2-pre-S2. Enhance ATX/ lysophosphatidic acid signal transduction, and then affect the physiological and pathological process of host cells.
【學(xué)位授予單位】：桂林醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R512.62

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