MicroRNA-520a通过失活AKT抑制HepG2.2.15细胞中HBV DNA复制

目的探讨microRNA-520a(miR-520a)抑制HepG2.2.15细胞中HBV DNA复制的机制。方法通过Entrez Nucleotide数据库预测AKT是否为miRNA-520a的靶基因。通过RT-qPCR和Western blot验证miRNA-520a是否调控HepG2.2.15细胞中AKT的表达,研究miRNA-520a模拟物及其抑制剂对HepG2.2.15细胞中HBV DNA转录和复制的影响。评价siRNA对AKT mRNA和蛋白表达以及HBV DNA转录和复制的作用。结果在HepG2.2.15细胞中,AKT为miRNA-520a的靶基因,miRNA-520a通过抑制AKT表达可进一步抑制HepG2.2.15细胞中HBV DNA的转录和复制。AKT表达下调受siRNAs诱导,同时抑制HepG2.2.15细胞中HBV的转录和复制。结论 MiRNA-520a可抑制AKT表达,进而抑制HBV DNA转录和复制,其可能成为治疗HBV感染的新靶点。     

组蛋白甲基转移酶SMYD3在不同HBV表达水平肝癌细胞中的差异表达

目的:探讨HBV组蛋白甲基转移酶SMYD3在肝癌细胞中的表达与HBV感染状态相关性.方法:选取HBV阴性和HBV阳性的肝癌细胞株HepG2、HepG2.2.15,利用实时逆转录聚合酶链反应(real time RT-PCR)方法检测 SMYD3mRNA表达水平,Western blot检测细胞中SMYD3蛋白表达差异.结果:SMYD3 mRNA及蛋白在HepG2.2.15的表达水平显著高于HepG2(0.92±0.12 vs 0.18±0.05,0.28±0.03 VS 0.54±0.05,均P<0.01),有统计学差异.结论:HBV可能通过上调SMYD3途径促进肝癌的恶性生物学行为.     

细胞内La蛋白对乙型肝炎病毒蛋白质表达的影响

目的 探讨细胞内La蛋白对HBV蛋白质表达的影响.方法 针对人La蛋白序列设计特异性的小分子干扰RNA(siRNA),通过体外转录的方法合成双链siRNA,并转染进入稳定表达HBV的HepG2.2.15细胞,实时荧光定量基因扩增技术测定HepG2.2.15细胞中的La蛋白mRNA变化水平;电化学发光分析技术检测HepG2.2.15细胞分泌的HBsAg和HBeAg变化情况.所得数据进行配对假设检验的统计学分析.结果 特异性siRNA干扰La蛋白mRNA的表达,使La蛋白在HepG2.2.15细胞中的表达降低;HepG2.2.15细胞分泌在培养上清液中的HBsAg和HBeAg表达显著下降,相关性分析显示,HBsAg、HBeAg变化水平与La蛋白mRNA变化水平呈正相关,相关系数分别为0.938和0.899.结论 特异性siRNA能够抑制细胞内La蛋白mRNA的表达,La蛋白可通过某种方式影响HBV蛋白质的表达.     

转录调节因子FoxO3a对小鼠Mac-1+细胞功能的影响

目的 探讨FoxO3a基因缺失对于小鼠Mac-1+细胞功能的影响.方法 采用流式细胞术计数FoxO3a基因敲除鼠及对照野生型鼠脾细胞中Mac-1+细胞数量及百分率.采用流式细胞分选技术分离脾细胞中Mac-1+细胞,利用Real-time PCR和Western印迹检测Mac-1+细胞中的炎性细胞因子S100A8、S100A9的表达水平.结果 FoxO3a基因敲除鼠的脾细胞中Mac-1+细胞数明显增加,细胞内S100A8和S100A9的基因及蛋白表达明显高于野生型鼠.结论 FoxO3a可能有抑制Mac-1+细胞过度活化、维持其稳态的作用.     

PTD-HBcAg融合蛋白诱导特异性CTL在HepG2.2.15细胞抑制HBV复制的研究

目的探讨PTD-HBcAg融合蛋白诱导小鼠体内特异性CTL并抑制HepG2.2.15细胞HBV复制的作用。方法 PTD-HBcAg、HBcAg和阴性对照分别与等体积的弗氏佐剂乳化后皮下免疫小鼠;第14d,分离脾淋巴细胞并分别用PTD-HBcAg、HBcAg、PTD和PBS加强刺激后收集上清,检测细胞因子IFN-γ、IL-12、IL-4和IL-10;刺激后的淋巴细胞作为效应细胞与HepG2.2.15细胞共培养,检测,效应细胞对HBsAg、HBVDNA的抑制作用及对HepG2.2.15细胞、HepG2细胞的杀伤效果。结果 PTD-HBcAg组分泌的IFN-γ、IL-12、IL-4和IL-10与HBcAg组和阴性对照组比较差异有统计学意义(P0.05);PTD-HBcAg融合蛋白组较HBcAg组和阴性对照组有更明显的病毒抑制作用(P0.05);PTD-HBcAg组对HepG2.2.15细胞的杀伤率明显高于HBcAg组和阴性对照组(P0.05)。结论 PTD-HBcAg可诱导HBV特异性CTL,能有效抑制HepG2.2.15细胞HBV的复制。     

SUMO特异性蛋白酶3在HBV复制中的作用机制

目的初步探讨SUMO特异性蛋白酶3(SENP3)在HBV感染中的作用机制。方法体外培养HepG2.2.15细胞,或利用HBV转基因小鼠肝脏组织,通过表达SiRNA干扰等,经蛋白质印迹、实时PCR、免疫组织化学染色等实验技术,检测SENP3在HBV感染细胞模型中的表达。结果 HBV DNA与其PBMC中SENP3的含量呈反比。健康对照组PBMC中SENP3的平均蛋白水平是CHB患者的7.4倍(P0.05)。HBV转基因小鼠肝脏中SENP3表达显著降低,小鼠肝脏组织中AKT磷酸化水平增高,是健康对照组的4.1倍(P0.01)。SENP3在HBV DNA全基因重组质粒转染的HepG2受体细胞(HepG2.2.15)中表达量显著低于未转染HBV DNA的HepG2细胞。与HepG2细胞相比,HepG2.2.15细胞中AKT磷酸化水平显著增高,其含量平均是HepG2细胞的5.2倍(P0.01);在HepG2.2.15细胞中转染SENP3质粒使其过表达,则AKT磷酸化水平降低,HBV DNA含量降低,并与SENP3质粒浓度呈负相关性;分别向HepG2.2.15细胞转染100ng和500ng的SENP3质粒,则细胞中HBV DNA含量由对照组中的7.983.03×106 IU/mL降低至3.45×106 IU/mL和1.92×106 IU/mL;应用siRNA干扰SENP3在HepG2.2.15细胞中的表达,则Akt磷酸化水平升高,其浓度是对照组的5.6倍(P0.01)。结论机体HBV DNA载量与SENP3的表达量呈负相关,SENP3可通过抑制肝细胞AKT磷酸化水平,降低HBV DNA载量。     

HBx基因对HepG2.2.15细胞MICA-A5.1表达及侵袭、迁移的影响

目的探究HBx基因对HepG2.2.15细胞侵袭、迁移及主要组织相容性复合体(MHC)Ⅰ类链相关基因A(MICA)-A5.1表达的影响。方法体外培养HepG2.2.15细胞系(插入HBV全基因组并持续表达的HepG2细胞),随机分为对照组、HBx过表达质粒组、HBx空载质粒组、HBx siRNA组、HBx siRNA阴性对照组,分别转染质粒及siRNA后,以CKK-8法分别检测24 h、48 h后细胞增殖情况,筛选合适药物作用时间;以Transwell侵袭实验和细胞划痕实验检测各组细胞侵袭迁移能力;以免疫印记法检测HBx、MICA-A5.1蛋白和上皮间质转化标志蛋白E-cadherin、Vimentin的表达;以酶联免疫吸附法检测各组细胞培养基中sMICA水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用SNK q检验。结果与对照组比较,24 h后,HBx过表达质粒组细胞活力升高,HBx siRNA组细胞活力降低,差异均有统计学意义(q值分别为8.268、4.365,P值分别为<0.001、0.036);48 h后,HBx过表达质粒组细胞活力升高,HBx siRNA组细胞活力降低,差异均有统计学意义(q值分别为12.680、7.523,P值均<0.001)。与对照组比较,HBx过表达质粒组细胞HBx、MICA及Vimentin蛋白表达、细胞培养基中sMICA水平、迁移细胞数目、侵出Transwell小室的细胞数目均升高,E-cadherin表达降低(q值分别为9.427、6.697、10.500、5.042、22.740、15.720、5.258,P值均<0.05);HBx siRNA组细胞HBx、MICA及Vimentin蛋白表达、细胞培养基中sMICA水平、迁移细胞数目、侵出Transwell小室的细胞数目均降低,E-cadherin表达升高(q值分别为8.133、8.828、7.616、7.673、5.391、7.694、6.226,P值均<0.05)。结论HBx基因调控HepG2.2.15细胞MICA-A5.1表达及侵袭、迁移,上调该基因可促进MICA-A5.1表达,增强HepG2.2.15细胞侵袭转移能力。     

靶向乙型肝炎病毒X区的siRNAs对HepG2.2.15细胞HBV复制的抑制作用

目的观察siRNA对HBV基因表达和复制的抑制情况。方法针对HBVX区设计并化学合成4条siRNAs,观察在不同浓度下对HepG2.2.15细胞HBV复制的抑制作用。结果 siRNA在30nmoL/L浓度时,4种siRNA对HepG2.2.15细胞HbsAg和HBeAg无明显的抑制作用(P0.05);在60nmoL/L和90nmoL/L浓度下,siR-NA-1和siRNA-4有明显的抑制作用(P0.05),他们对HBsAg和HBeAg的抑制率分别为41%和43%;siRNA能降低HepG2.2.15细胞HBVDNA的拷贝数。结论化学合成的siRNAs可以有效地抑制HBV基因的表达和复制。     

RNA干扰诱导肝癌HepG2细胞凋亡对丹参酮ⅡA药物敏感性的影响

目的:探讨载体介导的RNA干扰技术诱导HepG2细胞凋亡对丹参酮ⅡA药物敏感性的影响.方法:应用靶向细胞周期E(cyclin E)基因的siRNA真核表达载体转染HepG2细胞诱导凋亡,再以丹参酮ⅡA处理48h(cyclinE siRNA+丹参酮ⅡA组),同时设立cyclin EsiRNA组、无义siRNA组、丹参酮ⅡA组以及空白对照组,流式细胞仪检测细胞周期及凋亡率,吖啶橙荧光染色检测细胞凋亡形态,Western blot法观察细胞Caspase-3蛋白表达.结果:单用cyclinE siRNA质粒转染或丹参酮ⅡA处理肝癌HepG2细胞,细胞凋亡率显著高于空白对照组(P＜0.01),2μg/ml剂量凋亡率分别达到21.6%、23.6%.cyclinE siRNA质粒转染预诱导再经丹参酮ⅡA处理,HepG2细胞G0～G1期比例显著增高,S期、G2～M期细胞所占有比例明显下降;与单用cyclinE siRNA质粒转染、丹参酮ⅡA两组比较,细胞凋亡率分别增高了1.81和1.61倍,比两组合计增高0.35倍,Caspase-3蛋白表达水平分别增高了0.91倍和0.66倍.结论:通过cyclinE siRNA质粒转染诱导的HepG2细胞凋亡可提高丹参酮ⅡA药物敏感性,其机制与增强凋亡控制相关基因表达有关.     

HIF-2α基因沉默对人肝癌细胞株HepG2增殖能力的影响

目的探讨缺氧诱导因子-2α(hypoxia-inducible factors-2α,HIF-2α)表达水平对人肝癌细胞株HepG2增殖能力的影响。方法用氯化钴(CoCl_2)诱导细胞模拟缺氧微环境,并根据CoCl_2浓度不同分为50μmol/L、100μmol/L、200μmol/L、400μmol/L组,选择CoCl_2浓度为200μmol/L模拟肿瘤内缺氧微环境。采用siRNA干扰沉默HepG2细胞中HIF-2α的表达,分别应用RT-PCR、Western blotting方法检测HIF-2αmRNA和蛋白的表达,MTT方法检测HepG2细胞增殖,绘制细胞生长曲线,流式细胞技术观察细胞周期的改变。结果建立细胞缺氧模型,作为低氧组;采用浓度为50 nmol/L的HIF-2αsiRNA转染缺氧环境下的HepG2细胞,作为低氧+HIF-2αsiRNA组,结果显示:低氧组、低氧+Control siRNA组的HIF-2αmRNA和蛋白表达显著高于常氧组和低氧+HIF-2αsiRNA组(P0.05);细胞生长曲线结果显示:低氧组、低氧+Control siRNA组的光密度值显著高于常氧组和低氧+HIF-2αsiRNA组(P0.05);细胞周期结果显示:低氧组、低氧+Control siRNA组HepG2细胞在DNA合成前期(G0/G1期)细胞比率显著低于常氧组和低氧+HIF-2αsiRNA组,合成期(S)及合成后期(G2/M)细胞比率显著高于常氧组和低氧+HIF-2αsiRNA组(P0.05)。结论缺氧微环境通过促进人肝癌细胞株HepG2细胞中HIF-2α的表达进一步促进HepG2细胞增殖,而针对HIF-2α的靶向siRNA能有效抑制肝癌HepG2细胞中HIF-2α的表达,从而抑制HepG2细胞的增殖。     

Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells

AIM: To observe the inhibition of hepatitis B virus (HBV) replication and expression by combination of siRNA and lamivudine in HepG2.2.15 cells. METHODS: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. The transfected cells were cultured in lamivudine-containing medium (0.05 μmol/L) and harvested at 48, 72 and 96 h. The concentration of HBeAg and HBsAg was determined using ELISA. HBV DNA replication was examined by real- time PCR and the level of HBV mRNA was measured by RT-PCR. RESULTS: In HepG2.2.15 cells treated with combination of siRNA and lamivudine, the secretion of HBeAg and HBsAg into the supernatant was found to be inhibited by 91.80% and 82.40% (2.89 ± 0.48 vs 11.73 ± 0.38, P < 0.05; 4.59 ± 0.57 vs 16.25 ± 0.48, P < 0.05) at 96 h, respectively; the number of HBV DNA copies within culture medium was also significantly decreased at 96 h (1.04 ± 0.26 vs 8.35 ± 0.33, P < 0.05). Moreover, mRNA concentration in HepG2.2.15 cells treated with combination of siRNA and lamivudine was obviously lower compared to those treated either with siRNA or lamivudine (19.44 ± 0.17 vs 33.27 ± 0.21 or 79.9 ± 0.13, P < 0.05). CONCLUSION: Combination of siRNA and lamivudine is more effective in inhibiting HBV replication as compared to the single use of siRNA or lamivudine in HepG2.2.15 cells.     

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

INTRODUCTION Hepatitis B is a severe infectious disease threatening peoples’ health all over the world. There is still no efficient therapy to control HBV persistent replication, which may lead to the development of liver cirrhosis and hepatocellualar ca…     

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.     

Inhibition of human La protein by RNA interference downregulates hepatitis B virus mRNA in 2.2.15 cells

AIM: To investigate the role of human La protein in HBV mRNA expression. METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen (HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR. RESULTS: SEC products containing U6+1 snRNA promoter, and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+1-hLa833 was the most efficient, which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+1-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HBs and HBe mRNA levels were significantly decreased by 8- and 66-fold in U6+1-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs. CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease of HBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.     

干扰素α和胸腺五肽协同干预对HepG2.2.15细胞APOBEC3A和APOBEC3B mRNA表达的影响

目的探讨IFNα和胸腺五肽(TP5)协同干预对HepG2.2.15细胞载脂蛋白B mRNA编辑酶催化多肽3A(APOBEC3A)、APOBEC3B基因mRNA表达水平的影响。方法将HepG2.2.15细胞分为空白对照组、IFN处理组、TP5处理组和IFNα联合TP5组,在处理的12、24、48和72 h用实时荧光定量PCR方法检测细胞APOBEC3A、APOBEC3B基因mRNA表达水平。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果与空白对照组相比,IFNα、IFNα联合TP5分别处理12、24、48和72 h后,APOBEC3A mRNA的表达水平显著提高,差异均有统计学意义(P值均<0.001)。与IFNα处理组相比,4个时间点IFNα联合TP5组APOBEC3A mRNA的表达水平显著提高,差异均有统计学意义(P值<0.001)。TP5处理在各时间点对APOBEC3A mRNA表达水平的影响差异无统计学意义(P值均>0.05)。与对照组相比,所有处理组中APOBEC3B的mRNA表达水平差异无统计学意义(P值均>0.05)。结论IFNα联合TP5处理可显著上调HepG2.2.15细胞中APOBEC3A mRNA的表达水平。     

APOBEC3G体外抗乙型肝炎病毒的研究

目的探讨载脂蛋白B mRNA编辑酶催化多肽样蛋白3G（apolipoprotein B mRNA editing enzyme catalytic polypeptide like3G,APOBEC3G）（也称为CEM15）体外抗乙型肝炎病毒（HBV）的作用及其机制。方法脂质体转染pcDNA3.1 Human APOBEC3G-Myc-6Xhis、pcDAN3.1/His-C进入HepG2.2.15细胞,转染后,RT-PCR证实转染基因的表达,Western Blot证实蛋白的表达。通过ELISA方法检测细胞上清液中乙型肝炎表面抗原（HBsAg）及乙型肝炎e抗原（HBeAg）,RT-PCR分析APOBEC3G对HBV mRNA转录的影响。结果APOBEC3G基因与蛋白在HepG2.2.15细胞都有表达,与空质粒转染组相比,pcDNA3.1 Human APOBEC3G-Myc-6Xhis转染组HBsAg含量下降70.38%,HBeAg含量下降62.88%,未转质粒细胞为空白对照组。结论APOBEC3G在体外可以抑制HBV复制,可以作为一种新型的抗病毒制剂治疗乙肝病毒感染。     

RNA干扰技术用于抗乙型肝炎病毒的实验研究

目的 构建针对乙型肝炎病毒表面抗原(HBsAg)和核心抗原的小干扰RNA(siRNA)表达载体pSuper-C，观察其对HepG2 2．2．15细胞(简称2、2．15细胞)中HBV DNA转录和翻译相应蛋白的影响。方法 根据RNA干扰(RNAi)作用原理设计针对HBV核心区的相应序列，再将其克隆入含聚合酶ⅢH1-RNA启动子的真核表达载体pSuper，将此重组质粒以电转染法转入2．2．15细胞中，用酶联免疫吸附法(Abbott试剂)检测培养上清液中HBsAg和e抗原(HBeAg)的表达。结果 经酶切鉴定、电泳和测序分析证明，成功构建了含作用序列的重组质粒pSuper-C；但以电穿孔法转染 2．2．15细胞后末能发现其对2．2．15细胞培养上清液中的HBsAg和HBeAg的表达有影响。结论 RNAi在2．2．15细胞中的作用还需进一步的实验来证实。     

槲芪癥消汤对人肝癌细胞株HepG2.2.15增殖和凋亡的影响

目的:研究槲芪癥消汤对体外培养的人肝癌细胞株HepG2.2.15的增殖、凋亡、迁移能力、细胞内p53基因及蛋白表达量的影响,揭示该方可能的抗肿瘤机制。方法:应用不同浓度的槲芪癥消汤处理细胞,MTT法测定细胞增殖抑制率,Transwell检测细胞侵袭能力、流式细胞术检测细胞凋亡情况、rt-qPCR及Western blot检测细胞内p53的RNA及蛋白的表达变化。结果:槲芪癥消汤对HepG2.2.15细胞增殖有明显的抑制作用,使细胞的侵袭能力明显下降,并可促进细胞凋亡,同时细胞内p53的RNA及蛋白表达量明显增加,与对照组比较,差异均有统计学意义(均P<0.05)。结论:槲芪癥消汤能抑制肝癌HepG2.2.15细胞的增殖、侵袭,促进其凋亡,可能与促进细胞内的p53基因表达有关。