肝癌细胞清除乙型肝炎病毒的非免疫机制

目的 试图用肝癌细胞体外培养试验,推测肝细胞表达ＣＤ９５配体是否能自身通过凋亡来清除乙型肝炎病毒。方法 诱导ＨｅｐＧ２细胞表达ＣＤ９５Ｌ,与转染ＨＢＶ,表达ＣＤＳ９５的ＨｅｐＧ２．２．１５细胞共培养,以荧光显微镜和流式细胞仪检测后者的凋亡。结果 表达ＣＤ９５Ｌ的ＨｅｐＧ２细胞杀伤表达ＣＤ９５的ＨｅｐＧ２．２．１５细胞,２４小时凋亡１６．５％,４８小时凋亡４３．０％,这一作用可用抗－ＣＤ９５阻断。     

表达CD95L和HepG2细胞自分泌和旁分泌的细胞毒效应

阐明乙型肝炎时肝细胞可表达细胞效应分子ＣＤ９５Ｌ的效应机制。方法ＨｅｐＧ２细胞诱导表达ＣＤ９５Ｌ,与ＨｅｐＧ２．２．１５细胞共培养,或以其培养上清液加于另一份未经诱导的ＨｅｐＧ２细胞中,以流式细胞仪检测后者的凋亡率;以单个ＨｅｐＧ２细胞培养试验自体凋亡。     

表达CD95L的HepG_2细胞自分泌和旁分泌的细胞毒效应

目的阐明乙型肝炎时肝细胞可表达细胞毒效应分子ＣＤ９５Ｌ的效应机制。方法ＨｅｐＧ２细胞诱导表达ＣＤ９５Ｌ,与ＨｅｎＧ２．２．１５细胞共培养;或以其培养上清液加于另一份未经诱导的ＨｅＰＧｚ细胞中,以流式细胞仪检测后者的凋亡率;以单个ＨｅｐＧ２细胞培养试验自体凋亡。结果表达ＣＤ９５Ｌ的ＨｅｐＧ２细胞致表达ＣＤ９５的ＨｅｐＧ２．２．１５细胞的凋亡率为：２４小时时１６．５％,４８小时时４３．０％;培养上清液引起的凋亡率：２４小时时３８．７％,４８小时时７３．３％,单个ＨｅｐＧ２细胞经诱导的自杀率２４小时为４３．８％,均高于各自的对照。结论ＨｅｐＧｅ细胞以ＣＤ９５Ｌ的膜性或可溶性分子、经旁分泌或自分泌发生“自杀”或“同胞相杀”。     

感染乙型肝炎病毒MT2淋巴细胞表达Fas

目的：观测Ｔ淋巴细胞－ＭＴ２细胞对乙型肝炎病毒感染的敏感性，了解病毒感染对淋巴细胞表达Ｆａｓ的影响。方法：体外培养ＭＴ２细胞，用ＨＢＶ ＤＮＡ阳性血清直接感染，用酶联免疫和斑点杂交，ＰＣＲ法检测感染后的细胞病毒复制与表达状况，用酶联免疫法检测了Ｆａｓ抗原表达。结果：ＭＴ２细胞于阳性血清感染后第八天可测到ＨＢｅＡｇ，ＨＢｓＡｇ和ＨＢＶＤＮＡ同时出现Ｆａｓ表达信号，结论：ＭＴ２细胞对ＨＢＶ易感，ＨＢＶ     

转化生长因子β1正,反义基因对人肝癌细胞SMMC—7721增殖的影响

目的探讨人转化生长因子β１（ＴＧＦβ１）正、反义基因对人肝癌细胞 ＳＭＭＣ－７７２１增殖的影响。方法将ＴＧＦβ１正、反义基因分别导入ＳＭＭＣ－７７２１细胞,观察各转染细胞生长情况、凋亡特征及Ｆａｓ、ｐ５３的表达。结果与空质粒转染细胞相比,正义ＴＧＦβ１基因转染细胞生长速度减慢,出现明显的细胞凋亡形态学改变,３－羟基末端标记法（ＴＵＮＥＬ）为阳性,而反义基因转染细胞未发生上述改变,两者都可检测到ｐ５３信号,但未检测到Ｆａｓ表达。结论ＴＧＦβ１可通过诱导ＳＭＭＣ－７７２１凋亡而对其生长起负调控作用,其机制可能与Ｆａｓ／ＦａｓＬ、ｐ５３无关。     

慢性肝病患者肝组织Fas抗原的表达及意义

Ｆａｓ是多种细胞表面的一种膜蛋白，为Ｆａｓ配体（ＦａｓＬ）的受体，效应细胞的ＦａｓＬ与靶细胞的Ｆａｓ结合时，可诱导靶细胞凋亡［１］．有研究报道［２］，病毒性肝炎患者肝细胞的Ｆａｓ抗原表达与肝组织病理损伤相关，本研究旨在探讨慢性乙型、丙型病毒性肝炎、肝...     

凋亡相关基因Fas、Fas配体及bax在慢性病毒肝炎组织中的表达及意义

为探讨凋亡相关基因Ｆａｓ、Ｆａｓ配体及ｂａｘ在慢性病毒性肝炎中表达的意义。采用免疫组化技术研究４８例慢性肝炎（乙型肝炎３３例,丙型肝炎１５例）组织中Ｆａｓ、ＦａｓＬ及ｂａｘ的表达。结果：慢性乙型肝炎和丙型肝炎组织中Ｆａｓ、ＦａｓＬ及ｂａｘ表达均较正常肝增加,以细胞坏死和炎细胞浸润区域增加明显。结论：凋亡相关基因Ｆａｓ、Ｆａｓ及ｂａｘ可能参与了肝炎病毒致肝细胞的损伤过程。     

乙型肝炎病毒x蛋白对肝细胞FasL表达的激活作用

目的探讨ＨＢｘ蛋白对肝细胞Ｆａｓ配基（ＦａｓＬ）表达的调控作用。方法构建ＨＢｘ基因的真核表达载体ｐＣＥＰ４ｘ,电击法转染ＨｅｐＧ２细胞,潮霉素筛选阳性克隆细胞ＨｅＰＧ２Ｘ。用免疫细胞化学染色和免疫蛋白印迹法检测转染ＨＢｘ基因的ＨｅｎＧ２ｘ细胞ＦａｓＬ蛋白的表达。结果逆转录ＰＣＲ祛分析表明,ＨＢｘ基因在转染细胞内可有效转录;ＨｅｐＧ２ｘ细胞出现新的ＦａｓＬ蛋白表达。结论乙肝病毒ｘ蛋白可激活肝细胞表达ＦａｓＬ。     

三螺旋形成寡核苷酸抑制乙型肝炎病毒复制及抗原合成的实验研究

目的 探讨三螺旋形成寡核苷酸抗乙型肝炎病毒（ＨＢＶ）作用。方法 针对ＨＢＶ核心启动子ＳＰ１位点,合成２１ｍｅｒ硫代磷酸三螺旋形成寡核苷酸及２１ｍｅｒ无关对照寡核苷酸。采用ＬＥＩＳＡ,斑点杂交法分别检测了经寡核苷酸处理的ＨｅｐＧ２．２．１５细胞及空白对照组细胞培养上清ＨＢｓＡｇ,ＨＢｅＡｇ及ＨＢＶ ＤＮＡ水平。结果 ＴＦＯ２１组２．２．１５细胞ＨＢｓＡｇ及ＨＢＶ ＤＮＡ分泌量明显低于空白对照组。ＴＦ     

肝组织Fas抗原表达和细胞凋亡在乙型肝炎发病机理中的意义

目的：探讨肝组织中Ｆａｓ抗在表达和细胞凋亡的关系及其在乙型病毒性肝炎发病机理中的意义。方法：应用Ｆａｓ抗原免疫组织化学染色和原位末端标记技术（ＴＵＮＥＬ试验）。原位观察８４例乙型肝炎肝组织中Ｆａｓ抗原表达强度和分布及凋亡细胞密度。结果：８例正常肝组织未见有Ｆａｓ抗原表达,慢性重型肝炎的Ｆａｓ抗原阳性表达率为１００％,明显高于慢性肝炎中重度的６６．７％（Ｐ〈０．０１）;而慢性肝炎中重度的Ｆａｓ阳性率     

青蒿琥酯对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响

目的 探讨青蒿琥酯在体外对乙型肝炎病毒复制及肝癌细胞株HepG2.2.15凋亡的影响.方法 将不同浓度青蒿琥酯作用于转染乙型肝炎病毒全基因组DNA的肝癌细胞株HepG2.2.15,收集48 h上清,采用酶联免疫吸附实验检测上清中乙型肝炎病毒表面抗原(HBsAg)和e抗原(HBeAg),采用荧光定量PCR法检测HBV-DNA,流式细胞术检测细胞凋亡.结果 青蒿琥酯对HBV复制具有抑制作用,随着浓度增加,对HBsAg和HBeAg的抑制率逐渐上升,细胞内HBV-DNA 复制水平下降;青蒿琥酯可诱导肝癌细胞早期凋亡及导致细胞死亡,随浓度增加,HepG2.2.15细胞早期凋亡率及死亡率均增加.结论 青蒿琥酯对HepG2.2.15细胞HBsAg和HBeAg的分泌及HBV-DNA复制具有抑制作用,并具有诱导HepG2.2.15细胞凋亡的作用.     

Inhibition effect produced by dominant negative mutant fusion protein PreS2-TLM-ScFv-HBcDN on HBV replication in vitro

A mammalian expression vector comprised of the PreS2-TLM (translocation motif), a single-chain variable fragment (ScFv) that binds to hepatitis B surface antigen (HBsAg) and the EGFP gene was constructed. A stably transformed cell line that could express and secrete the fusion protein (PreS2-TLM-ScFv-EGFP) was established. HBsAg-positive HepG2.2.15 cells and HepG2 and HeLa cells were incubated with the supernatant of the transformed cell line cultures for evaluating the cellular permeability of PreS2-TLM-ScFv-EGFP. The location of the fusion protein PreS2-TLM-ScFv-EGFP in HepG2.2.15 cells was observed with immunofluorescence staining. EGFP was next replaced by a dominant negative mutant of the hepatitis B virus core gene (HBcDN) for producing fusion protein PreS2-TLM-ScFv-HBcDN, which was detected by western blot. The supernatant containing fusion protein PreS2-TLM-ScFv-HBcDN was added to the cultures of HepG2.2.15 cells, and the packaged hepatitis B virus (HBV) pregenomic RNA expression levels in the cells were measured using qRT-PCR. The results of the in vitro study indicated that the packaged HBV pregenomic RNA expression levels in HepG2.2.15 cells significantly decreased when these cells were exposed to the supernatant at the dose of 25% for 24, 48 and 72 h, or at the dose of 12.5% for 72 h.     

Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

Expression of Fas antigen in liver tissue of patients with chronic hepatitis B and C

OBJECTIVES: Cells infected with the hepatitis B or C virus can be eliminated by an immune response mediated by cytotoxic T cells. Recently, the Fas Ligand has been detected on the surface of cytotoxic T cells, and is thought to induce cells to apoptosis by adhering to the Fas antigen. METHODS: To evaluate the role of the Fas antigen and apoptosis in chronic hepatitis B or C, we used immunohistochemistry to study Fas antigen expression in liver samples obtained from 30 patients infected with chronic hepatitis B and 32 patients with chronic hepatitis C. RESULTS: In samples from patients with chronic hepatitis C and B, Fas antigen was mainly expressed in the cytoplasm (partly at the membrane) of hepatocytes, and these positive cells were detected especially at the periportal region near 'piecemeal necrosis'. According to Knodell's scoring system for the histological activity index, the scores of periportal inflammation and necrosis were higher in cases that were positive for Fas antigen than in cases that were negative for the antigen (n = 62, P < 0.001) and there was a positive correlation between these scores and the degree of Fas antigen expression (r = 0.621). There was also a positive correlation between the scores of intralobular inflammation and necrosis and the degree of Fas antigen expression (n = 62, r = 0.522, P = 0.001). Fas antigen was not detected in patients without hepatitis infection. CONCLUSIONS: These findings suggest that apoptosis of hepatocytes is induced via the Fas antigen and contributes to the elimination of infected cells.     

HepG2与HepG2．2．15细胞中P53表达及细胞生物学活性的差异

目的观察HBV对肝癌细胞生物学活性的影响，探讨HBV导致原发性肝癌发生的机制。方法用Western blot方法检测HepG2细胞以及稳定转染了HBV质粒的HepG2．2．15细胞中P53蛋白的表达情况，观察二种细胞某些生物学活性，如细胞周期、细胞凋亡的差异及细胞生长曲线以及裸鼠成瘤情况。结果HepG2．2．15细胞中P53蛋白表达水平及细胞凋亡率均高于HepG2细胞，细胞周期检测发现处于“S”期的细胞HepG2．2．15多于HepG2，细胞生长曲线显示其生长速度快于HepG2细胞，并且具有成瘤性。结论HBV在细胞内复制对细胞的凋亡与增殖均具有增强作用，其对细胞凋亡的促进作用可能通过使P53稳定性增强，进而使其活性增强。HBV感染的细胞存在凋亡与增殖的抗衡，当细胞增殖占优势时，则易于生成肿瘤。     

维甲酸诱导基因-I诱导干扰素的产生在乙型肝炎病毒感染清除中的作用

目的探讨维甲酸诱导基因-I(RIG-I)在诱导HBV感染IFN产生中的影响,以期对阐明乙型肝炎慢性化机制,寻找新的治疗靶点提供新的思路。方法以RIG-I表达载体flagRIG-l-ful1转染培养6孔板中的HepG2、HepG2.2.15,24h后用水疱性口炎病毒(VSV)感染细胞,然后在0、8、16和24h收集细胞及培养上清液,采用quantitive RT-PCR、Western印迹检测RIG-I、MDA5、IPS-1、ISG54等基因的表达,ELISA检测培养上清液中分泌的IFN-β水平。结果 HepG2.2.15细胞在VSV感染后IFN-β分泌水平(11.18±1.34)pg/mL明显低于HepG2细胞(275.50±22.97)pg/mL(P<0.01);通过质粒flagRIG-I-ful1转染高表达RIG-I后,HepG2.2.15分泌IFN-β的能力恢复至(548.78±57.99)pg/mL与HepG2细胞(532.10±39.34)pg/mL接近的水平(P=0.7013)。结论 HepG2.2.15细胞感染VSV后IFN产生障碍,高表达RIG-I后IFN表达水平得到恢复,提示RIG-I功能缺陷导致抗病毒免疫应答降低,RIG-I可能在HBV感染清除中起重要作用。     

Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2

AIM:To investigate the biological features of hepatitis B virus(HBV)-transfected HepG2.2.15 cells. METHODS:The cell ultrastructure,cell cycle and apoptosis,and the abilities of proliferation and invasion of HBV-transfected HepG2.2.15 and the parent HepG2 cells were examined by electron microscopy,flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trans-well assay.Oncogenicity of the two cell lines was compared via subcutaneous injection and orthotopic injection or implantation ...     

B7-H1在HBV感染导致肝细胞癌中的调控作用

目的探讨B7-H1（B7-homolog1）在乙型肝炎病毒（HBV）感染导致的肝细胞癌（HCC）中的作用机制。方法通过慢病毒lentivirus．B7．H1转染人正常肝细胞系L02、人肝癌细胞系HepG2、HepG2．2．15后,应用MTT法观察细胞活性,流式细胞仪检测细胞的凋亡情况,Westernblot技术检测蛋白B7-H1、Survivin蛋白的变化情况。结果抑制B7-H1表达水平后,通过MTT检测表明细胞系HepG2．2．15细胞活性比HepG2细胞、L02细胞活性降低,流式细胞仪检测凋亡增加,同时Westernblot显示Survivin蛋白的表达减少。结论通过慢病毒转染抑$1JB7．H1的表达可以促进细胞系HepG2．2．15凋亡,B7-H1可能是通过调控Survivin的变化影响HBV感染的肿瘤细胞的增殖与凋亡。