乙型肝炎病毒反式调节蛋白对autotaxin表达影响的研究内皮素

发布时间：2018-04-28 09:17

  本文选题：乙型肝炎病毒 + autotaxin　； 参考：《桂林医学院》2017年硕士论文

【摘要】：目的:乙型肝炎病毒(HBV)感染仍是全球性的主要健康问题之一。乙型肝炎及其并发症的发病机制需要进一步研究。本研究探讨HBV反式调节因子X蛋白(HBx)、前S2蛋白(pre-S2)对autotaxin(ATX)表达的影响及其意义,为阐明乙型肝炎及其并发症的发病机制提供实验依据。方法:PCR扩增ATX启动子序列和HBx基因、pre-S2基因,并分别克隆入pGL3-Enhancer和pcDNA3.1(+)中,构建ATX启动子重组荧光素酶报告基因载体p GL3-ATX和HBx、pre-S2重组真核表达载体pcDNA3.1(+)-HBx、pcDNA3.1(+)-pre-S2,将重组载体共转染HepG2细胞,通过荧光素酶活性检测HBx、pre-S2对ATX基因启动子的作用。构建稳定表达HBx的HepG2.HBx细胞及稳定表达pre-S2的HepG2.pre-S2细胞,Western blotting检测ATX蛋白的表达。结果:双酶切检测和DNA测序均证实重组载体pGL3-ATX和pcDNA3.1(+)-HBx的构建与设计相符。荧光素酶活性检测显示pGL3-ATX荧光素酶活性是空载体pGL3-basic的10倍(P0.001)。共转染结果显示,pcDNA3.1(+)-HBx+pGL3-ATX组、pcDNA3.1(+)-pre-S2+pGL3-ATX组的荧光素酶活性分别是空载体pcDNA3.1(+)+pGL3-ATX组的1.47倍(P0.001)和1.15倍(p0.001)。琼脂糖凝胶电泳检测结果显示HBx和pre-S2基因片段分别在HepG2细胞中稳定表达。Western blotting检测结果显示,HBV稳转细胞株HepG2.2.15细胞、稳定表达HBx的HepG2.HBx细胞和稳定表达pre-S2的HepG2.pre-S2细胞相较于普通HepG2细胞,其ATX蛋白的表达均增强。结论:1.克隆了HBx基因和ATX启动子序列,且ATX启动子序列具有启动子活性;2.构建了HBx重组真核表达载体pcDNA3.1(+)-HBx和ATX启动子重组荧光素酶报告基因载体pGL3-ATX;3.建立了分别稳定表达HBx和pre-S2基因片段的细胞HepG2.HBx和HepG2.pre-S2;4.HBx和pre-S2均可增强ATX启动子荧光素酶报告基因活性;5.ATX蛋白在普通肝癌HepG2细胞、稳定表达pre-S2基因片段的HepG2.pre-S2细胞、稳定表达HBx基因片段的HepG2.HBx细胞和HBV稳定表达株HepG2.2.15细胞中的相对表达量依次增强;6.HBx和pre-S2可激活ATX基因启动子,上调ATX的表达,提示HBV感染可通过其反式调节蛋白HBx、pre-S2上调ATX表达,增强ATX/溶血磷脂酸信号转导,进而影响宿主细胞生理和病理过程。
[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.
【学位授予单位】：桂林医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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