【作者】 刁志宏；

【导师】 侯金林； 朱幼芙；

【作者基本信息】 第一军医大学 ， 内科学， 2006， 博士

【摘要】 背景乙型肝炎病毒(hepatitis B virus，HBV)感染呈世界性流行，急慢性肝炎中的特征性病理改变，如肝小叶内的点状坏死和汇管区边缘的嗜酸性坏死的实质均为凋亡，表明肝细胞凋亡是病毒性肝炎的重要病理现象，与肝组织炎症的发生有密切联系。一贯认为凋亡是细胞主动的死亡过程，不引起炎症反应，但这与临床现象并不符合。可能只有在凋亡细胞迅速被吞噬而无毒性物质漏出时，才不引发炎症反应。如凋亡细胞数过多或吞噬细胞识别、吞噬能力存在障碍，细胞内容物释放，将导致炎症反应及组织损伤，这可能是某些疾病的重要发病机制。病毒感染与宿主细胞凋亡之间可能有多方面的关系，HBV蛋白可能是调控肝细胞凋亡的一个必要因子。因HBV的X蛋白有多种活性，能调控细胞内的多种信号转导路径，故一些学者首先研究其对肝细胞凋亡的作用。但不同学者的研究结果不同：X蛋白与P53结合抑制凋亡、而促使细胞的恶性转化；最近的研究还表明X蛋白抑制Caspase 3的活性、或通过上调SAPK／JNK信号抑制Fas介导的凋亡；但与肿瘤坏死因子α(tumor necrosis factor-alpha，TNFα)结合则提高凋亡的敏感性，或通过其反式激活强化凋亡。提示同一病毒蛋白在肝细胞的不同情况可起完全相反的作用，也促使我们思索HBV是否还有某一成份能较稳定抑制肝细胞凋亡。HBV有2个核壳的开放读框，其一指导合成21kDa的P21蛋白(hepatitis Bvirus c agent, HBcAg)，是病毒核壳的结构成份；另一产生25kDa的前C／C蛋白，(终产物hepatitis B virus e agent, HBeAg)是免疫的调节因子，与乙型肝炎的发病机制关系最为密切。其在内质网膜上截去信号肽后成为P22~e。P22~e的大部分进入内质网，水解除去羧基端的肽段形成17kDa的HBeAg后才能向细胞外分泌。小部分P22~e仍滞留于肝细胞浆内，长期以来对滞留的P22~e在感染中的作用不明。在对HBV前C／nt 1896终止密码子变异的研究中，发现细胞内的P22~e能与HBcAg形成杂合的核壳结构而抑制病毒颗粒的包装过程，故前C／C蛋白的缺失可导致病毒高复制。但我国的慢性HBV感染在血清HBe转换时出现的前C变异，常伴随病毒复制序列水平降低，这与其具有高复制能力似相矛盾。对此可能的解释是前C变异株诱导感染肝细胞凋亡促使病毒清除，与HBeAg抑制免疫而增强病毒复制符合，从而提出：P22~e蛋白是否有抑制肝细胞凋亡的作用?本实验室刘定燮博士在博士研究中，曾经阐明了表达P22~e蛋白的HepG2细胞有抑制甲氨喋呤，环孢素A等诱导凋亡的作用，但是对于抑制凋亡的分子途径仍未进一研究。我们发现P22~e的后一段序列(AA23-73)与Fas(接收凋亡信号的细胞表面分子)死亡区结合蛋白(FADD)的死亡效应区(DED)有23.5％的同源性。肝细胞通过FADD对TNFα敏感而诱导凋亡，已知有与DED氨基酸同源序列的Ⅱ型单纯疱疹病毒E8蛋白和传染性软疣病毒MC159蛋白，能抑制Fas抗体(一种促效抗体，agonist antibody)和TNF诱导的细胞凋亡，HBV的P22~e蛋白可能也有相同的作用，但迄今尚无研究报告。滞留的P22~e通过TNF-TNFL、Fas-FasL中哪一条途径抑制细胞凋亡，目前仍未见相关研究论文发表。Tai等发现丙肝病毒(hepatitis C virus, HCV)感染的肝脏和HCV核心转染的细胞中都有核转录因子κB(nuclear factorκB, NF-κB)的激活，HCV感染的肝脏较正常者更易见到NF-κB的核染色，HCV核心转染的细胞中NF-κB激活产生对TNF诱导凋亡的抵抗，用四氢吡咯二硫代氨基甲酸盐(PDTC)抑制NF-κB活化能使敏感性恢复，这些结果提示HCV感染可能通过激活NF-κB抑制凋亡，且HCV能逃避宿主的免疫监视使得病毒持续存在，从而可能形成肝癌。也有报道HBV的X蛋白可激活NF-κB或经NF-κB抑止HepG2凋亡。一贯认为NF-κB在肝胚胎瘤发展以及肝细胞再生时，是一个基本的、必需的“生存因子”；而且NF-κB还被证实是细胞在各种外界刺激作用下起主要调节作用。一些病毒诱导的宿主细胞凋亡反应与NF-κB激活有关，病毒感染后激活的NF-κB可能是激活天然免疫从而产生抗病毒活性的快速途径。NF-κB在肝脏病变中的作用究竟是通过TNF-TNFL还是Fas-FasL途径，目前仍未见相关研究。NF-κB在成熟B细胞和浆细胞中发现的这种蛋白能与免疫球蛋白κ轻链内含增强子的特异性序列结合，该序列由10个核苷酸组成(5-GGGACTTTCC-3)，命名为NF-κB。NF-κB广泛存在于真核生物中，是一个由复杂的多肽亚单位组成的蛋白家族。它作为信号传导途径中的枢纽与机体免疫，肿瘤的发生、发展，细胞凋亡的调节以及胚胎发育等重要事件有着密切联系，是一种重要的核转录因子。目前，对NF-κB的研究已成为一个非常引人关注的领域。关于细胞凋亡，目前国内外均侧重于对凋亡信号在细胞内转导过程的研究。通过激活或是抑制细胞内某一蛋白的表达，研究其在细胞凋亡某一途径中的作用，丰富和完善细胞凋亡的通路，更清楚地阐明肝病的发病机制。从分子水平阐明凋亡发生的机理，将是凋亡研究的方向以及热点。此项课题将研究在以TNFα为刺激信号的细胞凋亡过程中，HBVP22~e，NF-κB在此过程中的相互作用，进一步阐明HBV感染与免疫的某些基本问题。为此，本课题得到了国家自然科学基金支持。目的克隆乙型肝炎病毒P22~e基因，构建其真核表达载体pEGFP-C2HBVP22~e，以人肝细胞癌HepG2细胞系为研究模型，探讨在肝细胞凋亡过程中，HBVP22~e基因是否通过NF-κB的信号途径来抑制肝细胞凋亡的发生。方法1．以含1.2 copies HBV基因(adr亚型)p3.8Ⅱ质粒为模板，以PrimerCl：1873-1891 5’GGA ATTCATGTCCAAGCTGTGCCTTGGGT3’(插入EcoR I酶切位点)，PrimerC2：2454-2434 5’GCGGATCCCTAACATTGAGATTCCCGA 3’(插入BamH I酶切位点)为一对引物按常规方法进行PCR，获得HBVP22~e基因cDNA。2．利用通用T载体pMD18-T，将PCR获得的HBVP22~e基因cDNA进行克隆，获得克隆载体pMD18-THBVP22~e，进行EcoR I，BamH I双酶切鉴定及克隆测序，以保证获得HBVP22~e基因序列准确无误。3．将pMD18-THBVP22~e EcoR I，BamH I双酶切获得HBVP22~e cDNA回收纯化，利用pEGFP-C2质粒，构建HBVP22~e真核表达载体pEGFP-C2HBVP22~e。进行EcoR I，BamH I双酶切鉴定及测序，以保证以后表达HBVP22~e基因序列准确无误。4．脂质体转染pEGFP-C2HBVP22~e真核表达载体到HepG2细胞株中，经G418及有限稀释法筛选，获得含HBVP22~e基因的人肝细胞癌HepG2细胞株，并用免疫组化鉴定EGFP-HBVP22~e融合蛋白细胞内的表达；微粒子免疫检测培养上清中分泌的EGFP-HBVP22~e融合蛋白；荧光显微镜观察和Western Blot检测验证所获得的稳定转染细胞系能够稳定表达EGFP-HBVP22~e融合蛋白；Western Blot及细胞免疫组化使用抗HBe抗体验证融合了EGFP的HBP22~e蛋白的免疫原性。5．已终浓度333nM Act-D及100μg／L TNFα诱导刺激HepG2EGFP-HBVP22~e细胞凋亡，同时以HepG2细胞作为实验对照，刺激时间8h、16h、24h、32h、40h、48h，使用流式细胞术(Flow Cytometry, FCM)，利用碘化丙啶(Propidium iodide, PI)染色，检测具有亚G1划DNA含量的细胞比例，代表凋亡细胞数；使用激光共聚焦显微镜(Laser Scanning Confocal Microscopy)，观察Hoechst染色凋亡细胞核形态的变化。6．由TNFα诱导的HepG2EGFP-C2HBVP22~e细胞系的凋亡过程中，采用激光共聚交显微镜、经典的核蛋白电泳迁移率(electrophoretic mobility shift assay, EMSA)技术及采用NF-κB抑制剂Calpain InhibitorⅠ(ALLN)抑制其信号通路，检测观察NF-κB的核转移、活化等，以期探寻在HBVP22~e抑制肝细胞凋亡过程中，NF-κB信号途径的重要意义。结果1．经EcoR I，BamH I双酶切鉴定及克隆测序，证明获得HBVP22~e基因序列准确无误，获得HBVP22~e基因克隆载体pMD18-THBVP22~e。2．经EcoR I，BamH I双酶切鉴定及克隆测序，证明获得含HBVP22~e及增强型绿色荧光蛋白基因真核表达载体pEGFP-C2HBVP22~e。3．利用脂质体法转染HepG2细胞，用G418选择培养、有限稀释法筛选，荧光显微镜观察和Western Blot检测结果表明，所获得的稳定转染细胞系能够稳定表达EGFPHBVP22e融合蛋白。Western Blot及细胞免疫组化的阳性结果说明使用抗HBe抗体可以结合融合了EGFP的HBP22~e，融合蛋白中HBP22~e的抗原性没有发生改变。荧光显微镜观察和组化结果还可以看到HepG2中EGFPHBVP22~e的表达定位于胞浆与胞核，微粒子免疫荧光检测培养上清证实转染分泌蛋白含有HBeAg抗原性的物质。4．流式细胞术PI染色法检测以Act-D，TNFα诱导HepG2，HeoG2EGFP-C2HBVP22~e细胞系发生凋亡结果显示Oh，8h，16h两细胞系凋亡率凋亡比较，P＞0.05，差别不显著；而在24h，32h，40h，48h时，两细胞系凋亡率比较，P＜0.01，差别显著，HepG2EGFP-C2HBVP22~e细胞系凋亡率低于HepG2细胞系。激光共聚交显微镜观察两细胞系24h凋亡率比较，P＜0.01，差别显著。二者结果基本相符。5．激光共聚焦观察以Act-D，TNFα诱导HepG2，HepG2EGFP-C2HBVP22e细胞系发生凋亡前后NF-κB的核内外表达状况，结果显示HepG2EGFP-C2HBVP22~e诱导凋亡前、后，及诱导凋亡后的HepG2与未诱导凋亡的HepG2的NF-κB p65的核浆比例有显著性差异(χ~2=109.072，v=3，P=0.000，组间差异有显著性意义)；HepG2和HepG2EGFP-C2HBVP22~e凋亡后NF-κB p65的核浆比例有显著性差异(χ~2=44.298，v=1，P=0.000，2组间差异有显著性意义)，说明HepG2EGFP-C2HBVP22e细胞系发生凋亡前后NF-κB有明显的核迁移现象。6．HepG2，HepG2EGFP-C2HBVP22~e细胞系经ALLN处理后，流式细胞术检测以Act-D，TNFα诱导的凋亡率发生的变化，结果显示ALLN处理与否HepG2凋亡率相差不显著(t=0.544，P=0.615，差异没有统计学意义)。而ALLN处理HepG2EGFPC-2HBVP22~e后凋亡率显著高于未经ALLN处理的HepG2EGFPC-2HBVP22~e(t=7.515，P=0.002，差异有显著性意义)。说明ALLN抑制NF-κB，从而解除HBVP22~e对有凋亡的抑制作用。7．以Act-D，TNFα诱导HepG2，HepG2EGFP-C2HBVP22e细胞系发生凋亡后，EMSA分析NF-κB核转移状况显示HepG2凋亡前后NF-κB核迁移无差异(t=0.027，P=0.980，差异没有统计学意义)。而HepG2 EGFP-C2HBVP22~e凋亡前后NF-κB有明显的向核内迁移现象(t=9.431，P=0.001，差异有显著性意义)。结论1．实验结果表明，HepG2EGFP-C2HBVP22~e细胞系在发生凋亡的时间上比HepG2细胞系有明显延迟，及凋亡的发生率也明显低于HepG2细胞系，自此可证明HepG2EGFP-C2HBVP22~e细胞系中，由于外源性基因HBVP22~e的引入及表达，对细胞的凋亡有明显的抑制作用。2．激光共聚焦显微镜及EMSA观察以Act-D，TNFα诱导HepG2，HepG2EGFP-C2HBVP22~e细胞系发生凋亡前后NF-κB的核内外表达状况表明，HepG2EGFP-C2HBVP22~e细胞系发生凋亡前后，有明显的NF-κB向核内迁移现象。3．NF-κB抑制剂ALLN的使用，可以使以Act-D，TNFα诱导HepG2EGFP-C2HBVP22~e细胞系凋亡发生率升高。4．由上述结果可以初步推论出在HBVP22~e抑制肝细胞凋亡过程中，NF-κB信号途径有着重要意义。更多还原

【Abstract】 BACKGROUNDInfection of hepatitis B virus (HBV) is a very common disease in the world. The characteristic pathological changes of hepatitis, no matter acute or chronic, such as the spotty necrosis in hepatic lobules and the acidophilic necrosis, are in fact apoptosis. This fact shows that the apoptosis of hepatocytes is an important pathological phenomenon of viral hepatitis and is closely related to the inflammation of hepatic tissues.A widely accepted opinion on apoptosis is that apoptosis is the suicide of cell, and since it’s a death of cell activated by cell itself, it does not cause any inflammatory reaction. However, this opinion does not match with clinical observations. It is true that in some cases of apoptosis, there is not any inflammation observed, possibly because the destructed cells are engulfed by phagocytes rapidly and there is no leakage of poisonous substances. In other cases, when there is a extremely large number of destructed cells or the contents of cells are released, due to malfunction of phagocytes in recognizing and engulfing destructed cells, inflammatory reactions and damage of tissues can be seen and cause some diseases.It seems that there is a multiple relationship between viral infection and the cellular apoptosis of the host, and HBV protein may be a necessary factor regulating apoptosis of hepatocytes. Since the X protein of HBV has many activities and can modulate multiple signaling pathways in cells, many researchers select to study the role of HBV X protein on apoptosis. They came to different results. X protein can inhibit apoptosis and promote malignant transform of cell when it is bound with P53. Recent researches also showed that X protein can inhibit the activity of Caspase 3 and apoptosis induced by Fas by promoting SAPK/JNK signal. However, when bound with TNFα, X protein can increase the sensitivity of apoptosis or activate or promote apoptosis through other means. This suggests that the same viral protein may have different effects, depending on the situation of hepatocyte. In addition, it also suggests that there may be some factor in HBV that can stabilize or inhibit apoptosis of hepatocyte.HBV has an open reading frame with 2 core-shell structures. One of the core-shell structures can synthesize P21 protein (hepatitis B virus c agent, HBcAg) of 21kDa and is the structural component of viral core-shell structure. The other one produces pre-C/C protein of 25kDa, which final product is hepatitis B virus e agent (HBeAg), and is the immune modulating factor that is closely related to the mechanism of hepatitis B. After cutting off signal peptide on endoplasmic reticulum, it can turn to be P22~e. Most P22~e enter into endoplasmic reticulum and can only be released outside of cell after turning into HBeAg of 17kDa by removing peptide section on carboxyl end through hydrolysis. Some P22~e stay inside cytoplasm of hepatocyte and the role of such retained P22~e on infection remained unknown for a long time due to lack of research works on this area.According to the result of a research on the variation of pre-C/nt 1896 terminating codon of HBV, P22~e can combine with HBcAg and form a hybrid core-shell structure that can inhibit the packing process of viral particles. This result suggests that lack of pre-C/C protein may lead to high-level replication of virus. However, most chronic HBV infections in China show pre-C variation at the phase of serum HBe transformation and accompanying decrease of sequencing level of virus. It seems this fact conflicts with the high replication of virus. A possible explanation is that the pre-C mutant induces apoptosis of hepatocyte and promotes elimination of virus, and works with HBeAg to inhibit immune and promote the replication of virus. The question is: can P22~e inhibit apoptosis of hepatocyte? In his doctorate study, Dr. Liu Ding-xie, a co-researcher of our laboratory, has indicated that P22~e-expressing HepG2 can inhibit the apoptosis induced by Methotrexate and cyclosporine A. However, Dr. Liu did not make further study on the molecular pathway of this apoptosis inhibiting activity.We have found that there is a 23.5% homology between the latter sequence (AA23-73) of P22~e and the Death Effector Domain (DED) of Fas-associated death domain protein. FADD sensitizes to TNFαand induces apoptosis of hepatocyte. It is reported that the E8 protein of herpes simplex virus typeⅡand MC159 protein of molluscum contagiosum virus, both have homologous sequence of DED amino acids, can inhibit cellular apoptosis induced by Fas antibody (an agonist antibody) and TNF. It is possible that the P22~e protein of HBV also has similar effect. However, there is no report on this possibility. Which pathway does the retained P22~e use to inhibit cellular apoptosis? TNF-TNFL or Fas-FasL? Currently, there is not related report.According to the study of Tai and others, activation of nuclear factorκB (NF-κB) can be found in the liver infected by hepatitis C virus (HCV) and HCV core transfected cell; NF-κB nuclear staining is easier to be observed in HCV infected liver than in normal liver; activation of NF-κB in HCV core transinfected cell can inhibit TNF induced apoptosis; and the sensitivity can be recovered by inhibiting the activation of NF-κB using PDTC. These results suggest that HCV can inhibit apoptosis by activating NF-κB, and HCV can survive the immune monitoring process of the host and eventually cause hepatic carcinoma. Some reports indicate that HBV X protein can inhibit HepG2 apoptosis by activating NF-κB or through NF-κB. It has long been accepted that NF-κB is a fundamental and necessary "surviving factor" for the development of hepatic embryoma and regeneration of hepatocyte, and it is proved that NF-κB plays an important role in cell’s response to various stimuli. Some virus-induced apoptosis of host cell is related to the activation of NF-κB. It is possible that NF-κB is a fast pathway for activating natural immune and anti-virus activity. As for which pathway, TNF-TNFL or Fas-FasL, does NF-κB use to perform its role in the pathological changes of liver, currently there is not any related report.The protein found in mature B cell and plasma cell can bind with specific sequence of enhancer contained in the light chain of immunoglobulinκ. This sequence is consisted of 10 nucleotides (5-GGGACTTTCC-3) and is called NF-κB. Existing widely in eukaryotes, NF-κB is in fact a family of protein consisted of complicated subunits of polypeptides. It works as the hub of the route of signal transfer and is closely related with the occurrence and development of immune activity and tumor, the regulation of cellular apoptosis, embryonic development, and other important events of human body. It is also an important transcription factor. Currently, research on NF-κB has become an area of wide concern.Most researchers, no matter in China or in foreign countries, attach a higher priority to the study of the process of transfer of apoptosis signal. By activating or inhibiting expression of certain protein in cell, it is able to study the role of the protein in the process of cellular apoptosis, identify the routes of cellular apoptosis, and clarify the mechanism of liver diseases. The trend in this area is to clarify the mechanism of apoptosis from molecular level. Our research aims to study the interactions of HBV P22~e and NF-κB in the process of cellular apoptosis using TNFαas stimulating signal and further clarify some basic issues concerning HBV infection and immune reaction. This is why our research project is supported by State Fund of Natural Sciences.AIMHBV P22~e gene is cloned and its eucaryon expressing carrier pEGFP-C2HBVP22~e is constructed. Use human hepatocelluar carcinoma HepG2 cell lines to study whether HBV P22~e gene uses the signaling pathway of NF-κB to inhibit the occurrence of apoptosis of hepatocyte. METHODS1. Carry out PCR to obtain HBVP22~e gene cDNA using a template (p3.8Ⅱplasmid containing 1.2 copies HBV gene (adr subset)) and a pair of primers (Primer C1:1873-1891 5’GGA ATTCATGTCCAAGCTGTGCCTTGGGT3’ (inserted into EcoRⅠenzyme cutting site); Primer C2:2454-2434 5’GCGGATCCCTAACATTGAGATTCCCGA 3’ (inserted into BamHⅠenzyme cutting site)).2. After PCR, clone cDNA of HBVP22~e gene using general T-carrier pMD18-T to obtain cloned carrier pMD18—THBVP22~e. Carry out enzyme cutting verification and clone sequencing on EcoRⅠand BamHⅠto ensure that the sequence of HBVP22~e obtained is correct.3. Collect and purify HBVP22~e cDNA obtained through pMD18—THBVP22~e EcoRⅠand BamHⅠdouble enzyme cutting. Use pEGFP-C2 plasmid to construct pEGFP-C2HBVP22~e, the eukaryotic expression carder of HBVP22~e. Carry out FcoRⅠand BamHⅠenzyme cutting and sequencing to ensure the sequence of HBVP22~e gene be expressed correctly.4. Carry out liposome transfection to transfect eukaryotic expression carrier of pEGFP-C2HBVP22~e to HepG2 cell strains. Carry out G418 and limited dilution to obtain human hepatic cellular cancer strain HepG2 containing HBVP22~e gene and the expression of the gene in EGFP-HBVP22~e fusion protein is verified using immunohistochemical method. EGFP-HBVP22~e fusion protein secreted into supernatant is examined using corpuscle immune method. Fluorescence microscope observation and Western Blot are carried out to verify that stably transfected cell strains can express EGFP-HBVP22~e fusion protein consistently. The immunogenicity of HBP22~e protein is verified through Western Blot and immunohistochemical method using anti-HBe antibody.5. Stimulate and induce apoptosis of HepG2EGFP-HBVP22~e using 333nM Act-D and 100μg/L TNFα. HepG2 cells are used as control group. The stimulating duration is 8h, 16h, 24h, 32h, 40h, and 48h respectively. Carry out Flow Cytometry (FCM) and propidium iodide (PI) staining to examine the proportion of cells containing sub-G1 DNA. This proportion is used to represent the number of dead cells. Laser Scanning Confocal Microscopy and Hoechst staining are carried out to observe the change of the nucleus of dead cell.6. During the process of apoptosis of HepG2EGFP-C2HBVP22~e cell strain induced by TNFα, Laser Scanning Confocal Microscopy, traditional EMSA technique and NF-κB inhibitor ALLN are carried out or utilized to inhibit the signaling pathway. The nucleus transfer and activation of NF-κB are observed and the significance of NF-κB signaling pathway in HBVP22~e inhibition of apoptosis of hepatocyte is discussed.RESULTS1. The result of EcoRⅠand BamHⅠenzyme cutting and sequencing proved that the sequence of HBVP22~e gene is correct and the HBVP22~e clone carrier pMD18-THBVP22~e has been obtained successfully.2. The result of EcoRⅠand BamHⅠenzyme cutting and sequencing proved that HBVP22~e eukaryotic expression carrier, pEGFP-C2HBVP22~e, containing HBVP22~e and enhanced green fluorescent protein gene, has been obtained.3. HepG2 cells are transfected through liposome transfection. The cells are then selected and cultivated using G418 and screened using limited dilution method. The result of fluorescence microscope observation and Western Blot indicates that the stably transfected cell strains can express EGFPHBVP22~e fusion protein consistently. The positive results of Western Blot and immunohistochemical test indicate that anti-HBe antibody can combine with HBP22~e containing EGFP, and antigenicity of HBP22~e in fusion protein does not change at all. The result of fluorescence microscope observation and histochemical tests also indicate that the expressing location of EGFPHBVP22e in HepG2 is at cytoplasm and nucleus, and the result of microparticle immunoassay and Fluorescence Detection proved that there is substance with HBeAg antigenicity in the secreted transfected protein.4. The results of flow cytometry and PI staining show that for Act-D and TNFαinduced apoptosis of HepGw and HepG2EGFP-C2HBVP22~e cell strains, the difference of apoptosis rate of the two cell strains is not apparent at 0h, 8h, and 16h (P＞0.05). However, the apoptosis rates of the two cell strains showed significant difference at 24h, 32h, 40h, and 48h (P＜0.01), with the apoptosis rate of HepG2EGFP-C2HBVP22~e strain lower than that of HepG2 strain. The result of laser scanning confocal microscopy showed a significant difference of the apoptosis rate of the two cell strains at 24h (P＜0.01). Although different methods are used to examine the apoptosis rate, similar results are obtained.5. Laser scanning confocal microscopy is carried out to observe the expression of NF-κB inside and outside nucleus before and after Act-D and TNFαinduced apoptosis of HepG2 and HepG2EGFP-C2HBVP22~e strains. The results showed an apparent difference of NF-κB p65 content in nucleus and cytoplasm of HepG2EGFPC-2HBVP22~e cells before and after induced apoptosis, HepG2 cells after apoptosis, and HepG2 cells before apoptosis (x~2 =44.298, v=1, P=0.000, significant difference between groups). A significant difference of the content of NF-κB p65 in nucleus and cytoplasm in HepG2 and HepG2EGFP-C2HBVP22~e strains is also observed after apoptosis (x~2=44.298, v=1,P=0.100, significant difference between groups), indicating an apparent transfer of NF-κB to nucleus after apoptosis of HepG2EGFP-C2HBVP22~e strain.6. ALLN treatment is carried out on HepG2 and HepG2EGFP-C2HBVP22~e cell strains, and flow cytometry is carried out detect the change of the rate of apoptosis induced by Act-D and TNFα. The result shows that ALLN treatment is not related to the apoptosis rate of HepG2 strain (t=0.544, P=0.615, no statistical significance). However, the apoptosis rate of HepG2EGFPC-2HBVP22~e is significantly higher after ALLN treatment (t=7.515, P=0.002, significant difference), indicating that ALLN can inhibit NF-κB and reduce the apoptosis inhibiting effect of HBVP22.7. EMSA analysis is carried out to examine the nucleus transfer of NF-κB after Act-D and TNFαinduced apoptosis of HepG2 and HepG2EGFP-C2HBVP22ecell strains. The result shows no significant difference of nucleus transfer of NF-κB before and after apoptosis of HepG2 cells (t=0.027, P=0.980, no statistical significance). However, a significant transfer of NF-κB to nucleus is observed after apoptosis of HepG2 EGFP-C2HBVP22~e strain (t=9.431, P=0.001, significant difference).CONCLUSION1. The above results show that the apoptosis of HepG2EGFP-C2HBVP22~e strain occurs at a time much later than that of HepG2 strain, and its apoptosis rate is also apparently lower than that of HepG2 strain. This indicates that the introduction and expression of extraneous gene HBVP22~e significantly inhibits the apoptosis of HepG2EGFP-C2HBVP22~e strain.2. The result of laser scanning confocal microscopy and EMSA indicates an apparent transfer of NF-κB to nucleus after Act-D and TNFαinduced apoptosis of HepG2EGFP-C2HBVP22~e strain.3. NF-κB inhibitor ALLN can increase the rate of occurrence of Act-D and TNFαinduced apoptosis of HepG2EGFP-C2HBVP22~e strain.4. From the above results, it is able to draw a conclusion that NF-κB signaling pathway plays an important role in the activity of HBVP22~e in inhibiting apoptosis of hepatocyte.更多还原