HCV NS5A及其反式激活基因NS5ATP9对肝细胞凋亡的影响及机制研究

【作者】 赵崇山；

【导师】 赵龙凤；

【作者基本信息】 山西医科大学 ， 内科学， 2012， 硕士

【摘要】 第一部分HCV(Ib型)NS5A真核表达载体构建及凋亡相关基因表达谱芯片分析目的：构建HCV(Ib型)NS5A真核表达载体pcDNA3.1/myc-His(-)-HCV NS5A，研究NS5A蛋白对人肝癌细胞凋亡相关分子表达的影响。方法：以丙肝患者血清HCV RNA（Ib型）为模板，逆转录PCR法获取NS5A基因序列，再将该基因连到pGEM-T Easy载体上，经Xho I和Hind III双酶切获得NS5A粘性末端，将NS5A粘性末端与同样经Xho I和Hind III双酶切的pcDNA3.1/myc-His(-)连接从而构建成NS5A的真核表达载体。用Western blot法鉴定NS5A基因在人肝癌细胞系HepG2中的表达。将转染了pcDNA3.1/myc-His(-)-HCVNS5A的HepG2进行表达谱芯片分析。结果：构建的真核表达载体经双酶切及测序鉴定正确无误，转染HepG2细胞系后Western blot检测显示HepG2大量表达myc-his-NS5A融合蛋白。芯片分析显示11条与凋亡相关的基因出现差异表达其中5条基因表达上调，6条表达下调。结论：成功构建了Ib型HCV NS5A的真核表达载体，利用表达谱芯片分析发现NS5A对肝细胞凋亡的作用可能涉及死亡受体通路、线粒体凋亡通路、DNA修复机制。第二部分NS5ATP9抑制血清饥饿诱导的HepG2细胞凋亡目的：探讨HCV NS5A反式激活基因NS5ATP9对肝癌细胞系HepG2细胞凋亡的影响。方法：将NS5ATP9基因表达质粒、NS5ATP9干扰RNA质粒及各自的对照空质粒转染到HepG2细胞系，48 h后换无血清培养基培养24 h诱导凋亡，用实时荧光定量PCR验证转染后NS5ATP9 mRNA表达水平的变化，用Annexin V/7AAD流式细胞术检测细胞凋亡，JC-1检测线粒体膜电位，Western blot技术检测Bax蛋白表达，以观察NS5ATP9对HepG2细胞血清饥饿诱导凋亡的影响。结果：Annexin V/7AAD检测结果显示，NS5ATP9过表达组和对照组相比早期凋亡和总凋亡率显著减少（P<0.05）；而NS5ATP9干扰RNA组的早期凋亡、晚期凋亡和总凋亡率均显著增加（P<0.05）。JC-1检测结果显示，NS5ATP9过表达组和对照组相比线粒体膜电位保持正常形式的比例增加（P=0.053），而出现去极化电位形式的比例显著减少（P<0.05），Δψm有增高趋势(P=0.324)；而NS5ATP9干扰RNA组线粒体膜电位保持正常形式的比例显著减少（P<0.05），Δψm有降低趋势（P=0.378）。促凋亡分子Bax蛋白Western blot检测结果显示，NS5ATP9干扰RNA组Bax表达量显著上升（P<0.05），NS5ATP9过表达组Bax表达量则有下降趋势（P=0.551）。结论：NS5ATP9基因抑制血清饥饿诱导的HepG2凋亡，其机制可能是通过线粒体途径。更多还原

【Abstract】 Part IConstruction of eukaryotic expression vector of HCV(Ib) NS5A andanalysis of apoptosis-related genes via microarrayObjective To construct a recombinant expression plasmid carrying HCV NS5A(Ib) mediated bypcDNA3.1/myc-His(-) and explore its effect on apoptosis-related genes.Methods HCV NS5AcDNA was obtained from HCV RNA by reverse transcriptase PCR ,then NS5A was connected topGEM-T Easy vector。Both pGEM-T-HCV NS5A and pcDNA3.1/myc-His(-) were cut bydouble-enzyme cleavage method using Xho I and Hind III to make sticky ends .Then,weconnected pcDNA3.1/myc-His(-) with NS5A. The pcDNA3.1/myc-His(-)-HCV NS5A plasmidwas transfected to HepG2,then NS5A protein expression was detected by Wesern blot. RestultsThe recombinant eukaryotic expression vector was verified by double-enzyme cleavage methodand DNA sequencing analysis, NS5A protein expression was also confirmed by Wesern blot. Aset of 11 genes related apoptosis(6 up-regulated, 5 down-regulated) whose expression wasmodified by at least twofold was selected from oligonucleotide microarrays.Conclusion Wesucceeded in constructing NS5A recombinant eukaryotic expression vector. NS5A may promoteapoptosis of HepG2 through many pathway,including death receptor pathway, mitochondrialapoptosis pathway,DNA repairment.Part IINS5ATP9 expression inhibits the apoptosis of HepG2 induced byserum starvationObjective To investigate the effect of NS5ATP9 on the apoptosis of HepG2. Methods NS5ATP9exptession plasmid (pEGFP-N1-NS5ATP9) and miR RNAi expression vector forNS5ATP9(pcDNA6.2-GW/EmGFP-miR-NS5ATP9) were transiently transfected into HepG2cells by jet PRIME. Cell apoptosis was induced by starvation.The expression of NS5ATP9 inHepG2 was detected by semiquantitative RT-PCR.Cell apoptosis was evaluated by AnnexinV/7AAD, mitochondrial membrane potentials was detected by JC-1 staining. Bax protein wassemiquantidied by Western blotting. Restults After overexpression of NS5ATP9, the percentageof early and total apoptosis decreased significantly ,detected by Annexin V/7AAD(P<0.05),when we knocked down the expression of NS5ATP9 by RNAi expression vector transfection, thepercentage of early ,late and total apoptosis increased significantly(P<0.05) .Compared tocontrol group,the percentage of cells with polarization membrane potential showed a risingtedency after overexpression of NS5ATP9(P=0.053),meanwhile cells with depolarizationmembrane potential decreased significantly(P<0.05),thenΔψm increased ,but notsignificantly(P=0.324);after knocking down of NS5ATP9 , the percentage of cells withpolarization membrane potential dereased significantly (P<0.05),meanwhileΔψm tent todecline(P=0.378).Overexpression of NS5ATP9 downregulated the expression of pro-apoptoticBax(P=0.551),and knocking down of NS5ATP9 induced upregulating of Bax significantly(P<0.05) .Conclusion Our investigation suggested that NS5ATP9 inhibits apoptosis viamitochondrial apoptosis pathway in HepG2.更多还原