Inhibition of hepatitis B virus production by Boehmeria nivea root extract in HepG2 2.2.15 cells

AIM: To explore the anti-hepatitis B virus (HBV) effects of Boehmeria nivea (B. nivea) root extract (BNE) by using the HepG2 2.2.15 cell model system. METHODS: Hepatitis B surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), and HBV DNA were measured by using ELISA and real-time PCR, respectively. Viral DNA replication and RNA expression were determined by using Southern and Northern blot, respectively. RESULTS: In HepG2 2.2.15 cells, HBeAg (60%, P < 0.01) and particle-associated HBV DNA (> 99%, P < 0.01) secretion into supernatant were significantly inhibited by BNE at a dose of 100 mg/L, whereas the HBsAg was not inhibited. With different doses of BNE, the reduced HBeAg was correlated with the inhibition of HBV DNA. The anti-HBV effect of BNE was not caused by its cytotoxicity to cells or inhibition of viral DNA replication and RNA expression. CONCLUSION: BNE could effectively reduce the HBV production and its anti-HBV machinery might differ from the nucleoside analogues.

HepG2.2.15-derived exosomes facilitate the activation and fibrosis of hepatic stellate cells

BACKGROUND The role of exosomes derived from HepG2.2.15 cells,which express hepatitis B virus(HBV)-related proteins,in triggering the activation of LX2 liver stellate cells and promoting liver fibrosis and cell proliferation remains elusive.The focus was on comprehending the relationship and influence of differentially expressed microRNAs(DE-miRNAs)within these exosomes.AIM To elucidate the effect of exosomes derived from HepG2.2.15 cells on the activation of hepatic stellate cell(HSC)LX2 and the progression of liver fibrosis.METHODS Exosomes from HepG2.2.15 cells,which express HBV-related proteins,were isolated from parental HepG2 and WRL68 cells.Western blotting was used to confirm the presence of the exosomal marker protein CD9.The activation of HSCs was assessed using oil red staining,whereas DiI staining facilitated the observation of exosomal uptake by LX2 cells.Additionally,we evaluated LX2 cell proliferation and fibrosis marker expression using 5-ethynyl-2′-deoxyuracil staining and western blotting,respectively.DE-miRNAs were analyzed using DESeq2.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were used to annotate the target genes of DE-miRNAs.RESULTS Exosomes from HepG2.2.15 cells were found to induced activation and enhanced proliferation and fibrosis in LX2 cells.A total of 27 miRNAs were differentially expressed in exosomes from HepG2.2.15 cells.GO analysis indicated that these DE-miRNA target genes were associated with cell differentiation,intracellular signal transduction,negative regulation of apoptosis,extracellular exosomes,and RNA binding.KEGG pathway analysis highlighted ubiquitin-mediated proteolysis,the MAPK signaling pathway,viral carcinogenesis,and the toll-like receptor signaling pathway,among others,as enriched in these targets.CONCLUSION These findings suggest that exosomes from HepG2.2.15 cells play a substantial role in the activation,proliferation,and fibrosis of LX2 cells and that DE-miRNAs within these exosomes contribute to the underlying mechanisms.

Evaluation of the intracellular lipid-lowering effect of polyphenols extract from highland barley in HepG2 cells

Active ingredients from highland barley have received considerable attention as natural products for developing treatments and dietary supplements against obesity.In practical application,the research of food combinations is more significant than a specific food component.This study investigated the lipid-lowering effect of highland barley polyphenols via lipase assay in vitro and HepG2 cells induced by oleic acid(OA).Five indexes,triglyceride(TG),total cholesterol(T-CHO),low density lipoprotein-cholesterol(LDL-C),aspartate aminotransferase(AST),and alanine aminotransferase(ALT),were used to evaluate the lipidlowering effect of highland barley extract.We also preliminary studied the lipid-lowering mechanism by Realtime fluorescent quantitative polymerase chain reaction(q PCR).The results indicated that highland barley extract contains many components with lipid-lowering effects,such as hyperoside and scoparone.In vitro,the lipase assay showed an 18.4%lipase inhibition rate when the additive contents of highland barley extract were 100μg/m L.The intracellular lipid-lowering effect of highland barley extract was examined using 0.25 mmol/L OA-induced HepG2 cells.The results showed that intracellular TG,LDL-C,and T-CHO content decreased by 34.4%,51.2%,and 18.4%,respectively.ALT and AST decreased by 51.6%and 20.7%compared with the untreated hyperlipidemic HepG2 cells.q PCR results showed that highland barley polyphenols could up-regulation the expression of lipid metabolism-related genes such as PPARγand Fabp4.

Reduction of the oxidative damage to H_(2)O_(2)-induced HepG2 cells via the Nrf2 signalling pathway by plant flavonoids Quercetin and Hyperoside

Hyperoside and quercetin are similar in molecular structures.In this study,the antioxidant regulatory targets of hyperoside and quercetin are mainly in the nuclear factor(erythroid-2-derived)-related factor 2(Nrf2)pathway predicted by network pharmacology.And the antioxidant effect and mechanism of hyperoside and quercetin were measured and compared in H_(2)O_(2)-induced Hep G2 cells and Caenorhabditis elegans.The findings indicated that quercetin was more effective than hyperoside in reducing oxidative damage,which was proved by improved cell viability,decreased reactive oxygen species(ROS)production,decreased cellular apoptosis,and alleviated mitochondrial damage.In addition,quercetin was more efficient than hyperoside in enhancing the expression of Nrf2-associated m RNAs,increasing the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT),and reducing the cellular malondialdehyde(MDA)content.Quercetin was superior to hyperoside in prolonging the lifespan of worms,decreasing the accumulation of lipofuscin,inhibiting ROS production,and increasing the proportion of skn-1 in the nucleus.With the Nrf2 inhibitor ML385,we verified that quercetin and hyperoside primarily protected the cells against oxidative damage via the Nrf2 signalling pathway.Furthermore,molecular docking and dynamics simulations demonstrated that the quercetin-Kelch-like ECH-associated protein 1(Keap1)complex was more stable than the hyperoside-Keap1 complex.The stable structure of the complex might hinder the binding of Nrf2 and Keap1 to release Nrf2 and facilitate its entry into the nucleus to play an antioxidant role.Overall,quercetin had a better antioxidant than hyperoside.

Effect of IL-18 on peripheral blood mononuclear cells of chronic hepatitis B and hepatitis B virus DNA released by HepG2.2.15 cell lines

BACKGROUND: Interleukin-18 (IL-18), a pro-inflamma- tory cytokine that induces interferon-γ (IFN-γ) production in T cells and natural killer cells, plays a critical role in the T-lymphocyte helper type 1 ( Th1) response. This study was designed to explore the effect of IL-18 on peripheral blood mononuclear cells ( PBMCs) derived from chronic hepatitis B (CHB) and on hepatitis B virus (HBV) DNA released by HepG2.2.15 cell lines, which were transfected with hepatitis B virus gene in vitro. METHODS: PBMCs isolated from 25 healthy people and 25 patients with CHB were stimulated with HBcAg and IL-18 of various concentrations for 72 hours. The levels of IFN-γ in the supernatants of cultured PBMCs were determined by ELISA. After the stimulation of IL-18 of various concentra- tions, PBMCs derived from one patient were co-cultured for 96 hours with HepG2. 2. 15 cells which had been cul- tured for 24 hours, and then the supernatants were collected by centrifugation and used for HBV DNA quantitative as- say. RESULTS: When PBMCs were stimulated by HBcAg and IL-18 at various concentrations, the levels of IFN-γ in the supernatants of CHB groups were much higher than those in normal control groups, at 0.2 ng/ml: t =11.70, P< 0.01; at 1.0 ng/ml: t =16.19, P<0.01; and at5.0 ng/ml: t =20.12, P <0.01. In the CHB groups, the levels of IFN-γ in the supernatants of PBMCs stimulated by HBcAg alone were lower than both those stimulated by HBcAg and EL-18 at various concentrations and those stimulated by HBcAg and EL-18 (5.0 ng/ml) together with EL-12 (mild: t = 2.20, P<0.05; moderate; t=2.97, P<0.05; severe; t = 0.66, P >0.05). The content of HBV DNA in the superna- tant of co-cultivation of HepG2. 2. 15 cells and PBMCs without stimulated materials was higher than that stimula-ted by HBcAg and EL-18 at various concentrations of HBc- Ag and IL-18 together with IL-12/IFN-α1lb. CONCLUSION: DL-18 can induce IFN-γ secretion and pro- bably play a key role in the modulation of both innate and adaptive immunity. It has implications in improving im- munoregulatory effect and increasing the ability of immune cells to kill cells infected by virus.

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay （ELISA）. Intracellular viral DNA and covalently closed circular DNA （cccDNA） were quantified by real-time polymerase chain reaction （PCR）. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR （RT-PCR）. RESULTS： siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION： Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.

Protective effect of brain and muscle arnt-like protein-1 against ethanol-induced ferroptosis by activating Nrf2 in mice liver and HepG2 cells

Alcohol abuse has recently become a serious health concern worldwide,and the incidence of alcoholic liver disease(ALD)is rapidly increasing with high morbidity and mortality.Ferroptosis is a newly recognized form of regulated cell death caused by the iron-dependent accumulation of lipid peroxidation.Here we showed that the circadian clock protein brain and muscle arnt-like protein-1(BMAL1)in hepatocytes is both necessary and sufficient to protect against ALD by mitigating ferroptosis.U pon exposure to alcohol(5%Lieber-DeCarli liquid alcohol diet for 10 days before binged alcohol with 5 g/kg body weight in vivo,300 mmol/L for 12 h in vitro,respectively),the content of iron,reactive oxygen species(ROS)and malondialdehyde(MDA)was boosted signifi cantly while glutathione(GSH)was decreased that mainly based on the downregulated protein expression of ferritin heavy chain(FTH),ferroportin(FPN),heme oxygenase1(HO-1)and anti-cystine/glutamate antiporter(SLC7A11),while these changes could be abolished by ferroptosis inhibitor Ferrostatin-1[Fer-1(5 mg/kg body weight for 10 days in vivo,10μmol/L for 2 h in vitro,respectively)].Further study indicated that the alcohol could activate the protein expression of BMAL1 which exerts a protective effect against ferroptosis through promoting nuclear factor erythroid 2-related factor 2(Nrf2)translocation into nuclear and subsequently stimulating its downstream proteins FTH,FPN,glutathione peroxidase 4 activity(GPX4),HO-1,SLC7A11,while knockdown of BMAL1 and Nrf2 by RNA interference further downregulated the expression of these protein and thus promoting ferroptosis in response to alcohol.Collectively,our results unveiled that the protective action of BMAL1 during alcohol challenge depends on its ability to activate Nrf2-ARE antiferroptosis pathway and targeting hepatic BMAL1 to dampen hepatic ferroptosis signaling may have therapeutic potential for ALD.

mRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles

BACKGROUND The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy.However,the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles(AgNPs)makes its precise mechanisms of action unclear.AIM To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G.Don(C.roseus)-AgNPs.METHODS The proliferative activity of hepatocellular carcinoma(HepG2)and normal human liver(THLE3)cells treated with C.roseusAgNPs were measured using MTT assay.The RNA samples were extracted and sequenced using BGIseq500 platform.This is followed by data filtering,mapping,gene expression analysis,differentially expression genes analysis,Gene Ontology analysis,and pathway analysis.RESULTS The mean IC 50 values of C.roseusAgNPs on HepG2 was 4.38±1.59μg/mL while on THLE3 cells was 800±1.55μg/mL.Transcriptome profiling revealed an alteration of 296 genes.C.roseusAgNPs induced the expression of stress-associated genes such as MT,HSP and HMOX-1.Cellular signalling pathways were potentially activated through MAPK,TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest.The alteration of ARF6,EHD2,FGFR3,RhoA,EEA1,VPS28,VPS25,and TSG101 indicated the uptake of C.roseus-AgNPs via both clathrin-dependent and clathrinindependent endocytosis.CONCLUSION This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells.The cytotoxicity effect is mediated by the aberrant gene alteration,and more interestingly the unique selective antiproliferative properties indicate the C.roseusAgNPs as an ideal anticancer candidate.

Interleukin-10 Is Expressed in HepG2.2.15 Cells and Regulated by STAT1 Pathway

This study investigated the expression profiles of IL-10 gene in three human hepatoma cell lines including Huh7, HepG2, and HepG2 transfected with a plasmid containing hepatitis B virus （HBV） named HepG2.2.15. RT-PCR analysis demonstrated that IL-10 message RNA was absent in HepG2 and Huh7 cells, whereas it was present in HepG2.2.15 cells, which was consistent with ELISA result. Furthermore, except for lamivudine other antiviral treatments did not significantly decrease the HBV DNA level in HepG2.2.15 cells, while they had different effects on the expression of IL-10 protein, although stimulation by LPS had no significant effect. In addition, except for poly（I：C）, the other treatments decreased the expression of IL-10 protein to different degrees, but had no sig-nificant effects on the expression of NF-κB and MyD88. Meanwhile, all treatments we used had effect on the expression of STAT1. In conclusion, IL-10 was expressed in HepG2.2.15 cells and STAT1 pathway might be involved in the regulation of IL-10 expression in HepG2.2.15 cells, but it was not the sole pathway, the exact mechanism warrants further study.

Anti-diabetic potential of apigenin,luteolin,and baicalein via partially activating PI3K/Akt/GLUT-4 signaling pathways in insulin-resistant HepG2 cells

Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities.In this study,apigenin,luteolin,and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant(IR)HepG2 cells.All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4(GLUT4)and phosphor-glycogen synthase kinase(GSK-3β).These fl avonoids signifi cantly inhibited the production of reactive oxygen species(ROS)and advanced glycation end-products(AGEs),which were closely related to the suppression of the phosphorylation form of NF-κB and P65.The expression levels of insulin receptor substrate-1(IRS-1),insulin receptor substrate-2(IRS-2)and phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway in IR-HepG2 cells were all partially activated by the fl avonoids,with variable effects.Furthermore,the intracellular metabolic conditions of the fl avonoids were also evaluated.

Naringin ameliorates H_(2)O_(2)-induced oxidative damage in cells and prolongs the lifespan of female Drosophila melanogaster via the insulin signaling pathway

Naringin exists in a wide range of Chinese herbal medicine and has proven to possess several pharmacological properties.In this study,PC12,HepG2 cells,and female Drosophila melanogaster were used to investigate the antioxidative and anti-aging effects of naringin and explore the underlying mechanisms.The results showed that naringin inhibited H_(2)O_(2)-induced decline in cell viability and decreased,the content of reactive oxygen species in cells.Meanwhile,naringin prolonged the lifespan of flies,enhanced the abilities of climbing and the resistance to stress,improved the activities of antioxidant enzymes,and decreased malondialdehyde content.Naringin also improved intestinal barrier dysfunction and reduced abnormal proliferation of intestinal stem cells.Moreover,naringin down-regulated the mRNA expressions of inr,chico,pi 3k,and akt-1,and up-regulated the mRNA expressions of dilp2,dilp3,dilp5,and foxo,thereby activating autophagy-related genes and increasing the number of lysosomes.Furthermore,the mutant stocks assays and computer molecular simulation results further indicated that naringin delayed aging by inhibiting the insulin signaling(IIS)pathway and activating the autophagy pathway,which was consistent with the result of network pharmacological predictions.

Ghrelin regulates insulin resistance by targeting insulin-like growth factor-1 receptor via miR-455-5p in hepatic cells

Objective: To explore the mechanism by which ghrelin regulates insulin sensitivity through modulation of miR-455-5p in hepatic cells. Methods: HepG2 cells were treated with or without DAG (1 μM). Glucose consumption, intracellular glycogen content, phosphorylation of PI3K and Akt stimulated by insulin, expression of miR-455-5p, as well as IGF-1R protein level were analyzed. In addition, bioinformatic analysis, dual luciferase reporter assay, miR- 455-5p mimic or inhibitor treatment was conducted to investigate the molecular mechanisms. Results: High glucose treatment upregulated miR-455-5p expression but reduced glucose consumption and glycogen content. DAG reversed the effect of high glucose on glucose metabolism, increased protein level of IGF-1R and phosphorylation of PI3K/Akt stimulated by insulin, as well as downregulated miR-455-5p expression. Bioinformatic analysis indicated IGF-1R was the target of miR-455-5p. Dual luciferase reporter assay, as well as transfection with miR-455-5p mimic/inhibitor confirmed that DAG activated IGF-1R/PI3K/Akt signaling via inhibiting miR-455-5p. Conclusion: DAG improves insulin resistance via miR-455-5p- mediated activation of IGF-1R/PI3K/Akt system, suggesting that suppression of miR-455-5p or activation of DAG may be potential targets for T2DM therapy.

荔枝核提取物对HepG2.2.15细胞系HBsAg与HBeAg表达的影响

目的 :研究荔枝核对乙型肝炎病毒的抑制作用及其有效部位。方法 :应用HepG 2 .2 .15细胞系培养系统检测荔枝核提取物A、B、C、D、E、F对HBsAg与HbeAg表达的影响。 结果 :荔枝核提取物A、B、C、D、E、F (2 0 0 ,10 0mg·L-1)对HBsAg和HbeAg表达均有抑制作用 ,其中E成分作用最强 ,在 2 0 0mg·L-1的浓度下 ,于实验第 3天对HBsAg的抑制率为 5 0 % ,对HBeAg的抑制率为 2 0 % ,于实验第 9天对HBsAg的抑制率为90 .9% ,对HBeAg的抑制率为84 .3% (与对照组比较P <0 .0 1)。结论 :荔枝核提取物体外有较强的抗乙肝病毒作用。

青钱柳提取物对HepG2 2.2.15细胞HBsAg和HBeAg表达的影响

目的研究青钱柳提取物体外抗乙肝病毒作用。方法青钱柳用75%乙醇提取后按溶剂极性萃取,获得的三氯甲烷部位经柱层析得到Fr.2组分。体外培养HepG2 2.2.15细胞,MTT法检测青钱柳提取物Fr.2组分对HepG22.2.15细胞的细胞毒作用,用ELISA法检测HepG2 2.2.15细胞培养液中HBsAg和HBeAg的表达。结果青钱柳提取物Fr.2组分对HepG2 2.2.15细胞无明显的细胞毒作用。与对照组比较,Fr.2组分对HBsAg和HBeAg的表达均有显著性抑制作用(P<0.01或0.05)。结论青钱柳提取物体外具有较强的抗乙肝病毒作用。

甘草甜素对HBsAg低表达HepG2.2.15细胞株HBV感染及TLR2、4的影响

目的:探讨甘草甜素(GL)对HepG2.2.15细胞上清HBV DNA、e抗原分泌,Toll样受体2、4(TLR2、4)信号分子的表达及对细胞增殖的影响。方法:实时荧光定量PCR检测HBV DNA表达,ELISA检测HBV所分泌抗原,MTT检测细胞增殖活性,直接免疫荧光流式细胞术(FCM)检测TLR2、4在细胞株上表达的阳性细胞率,并与空白对照组对比。结果:HBsAg在该细胞株低表达,但HBeAg则明显阳性,因此研究了GL对e抗原的影响,给药后第3d e抗原总体均数间差异显著(P<0.01),但只有400μg/ml组与对照组间比较显著降低(P<0.05),800μg/ml组则较其余GL组明显升高(P<0.01)。HBV DNA给药后第3 d总体均数间比较差异有显著性,只有50μg/ml组较对照组降低,但无统计学意义(P>0.05),其余各组均较对照组升高;各组TLR2、4数值总体均数间有显著差异(P<0.01)。除200μg/ml组外,各剂量组与对照组比较显示两者均显著上调(P<0.05),但均无剂量依赖关系;MTT实验显示200μg/ml以下三个剂量组均可促进细胞增殖,但只有200μg/ml组与对照组间差异显著(P<0.05);400、800μg/ml两组均显著抑制细胞增殖(P<0.01);而MTT分别与HBeAg、HBV DNA呈显著负相关(P<0.05)。结论:研究表明,GL对HepG2.2.15变异株的病毒复制及e抗原分泌可能具有双向作用;细胞的存活数与病毒复制及e抗原呈负相关;TLR2在变异株低表达,GL呈非剂量依赖关系上调TLR2、4表达,至少在该细胞株GL影响HBV的机理与TLR2、4信号的改变无关,但有可能在体内通过免疫途径影响HBV。

复方六月雪对HepG2.2.15细胞HBsAg和HBeAg的抑制作用

目的:观察复方六月雪(CLYX)体外抗乙型肝炎病毒(HBV)的作用。方法:采用四甲基噻唑蓝(MTT)法检测CLYX对HepG2.2.15细胞的半数毒性浓度(TC50)和最大无毒浓度(TC0);在TC0基础上观察不同浓度药物作用于HepG2.2.15细胞,分别在第72h和144h收集细胞培养上清液,采用酶联免疫吸附实验(ELISA)法测定上清液HBsAg和HBeAg的滴度。结果:TC50为3.070g·L-1,TC0为0.945g·L-1,复方六月雪对HepG2.2.15细胞毒性较低。无毒浓度的复方六月雪在HepG2.2.15细胞培养中可有效地抑制细胞HBsAg(乙型肝炎表面抗原)和HBeAg(乙型肝炎E抗原)的分泌;且治疗指数(TI)均大于2,为高效低毒的抗HBV药物。结论:CLYX在体外有显著的抗HBV的作用,且毒性较低。

恩替卡韦和干扰素序贯作用对HepG2.2.15细胞HBV DNA复制的影响

目的探讨恩替卡韦(Entecavir,ETV)与干扰素(IFNα-2b)序贯作用对Hep G2.2.15细胞HBV DNA复制的影响。方法将培养Hep G2.2.15细胞分组,单独组中分别加入不同浓度ETV的配制液(1.5μg/ml、5μg/ml、10μg/ml)或IFNα-2b配制液(5 000 IU/ml、10 000 IU/ml、15 000 IU/ml);联合组中联合加入ETV和IFNα-2b配制液;序贯组中先后加入ETV和IFNα-2b序贯配制液。在不同时点,采用MTT法检测Hep G2.2.15细胞增殖情况,采用实时荧光定量法检测各组细胞培养上清液中HBV DNA水平,酶联免疫法(ELISA)检测上清液中HBs Ag与HBe Ag水平。结果随着时间延长,序贯组对Hep G2.2.15的HBV DNA复制抑制作用明显大于其他两组(P<0.05);对HBs Ag与HBe Ag的抑制作用随时间的延长而加强(P<0.05)。结论 ETV与IFNα-2b序贯作用于Hep G2.2.15细胞,对Hep G2.2.15细胞HBV DNA复制有明显的抑制作用,且作用强于单独或联合使用。

基因芯片筛选HepG2与HepG2.2.15细胞中的差异表达基因

目的探讨HepG2细胞及HepG2.2.15细胞中差异表达的基因并对其基因表达谱进行生物学信息分析。方法用Trizol一步法提取HepG2细胞及HepG2.2.15细胞的总RNA,并纯化mRNA,反转录合成荧光分子(Cy3/Cy5)标记的cD-NA探针,与基因芯片杂交;采用LuxScan3.0图像分析软件对芯片图像进行分析,把图像信号转化为数字信号,最后以差异为2倍的标准来确定差异表达基因。结果在54614个基因表达谱的筛选中,发现有4462个基因表达水平显著上调,2592个基因表达水平显著下调。结论HBV基因组及其表达产物对于肝细胞基因表达谱有显著影响,可能参与了肝癌的发生发展。

甘草甜素对HepG2.2.15细胞株HBeAg水平及TLR4表达的影响

目的探讨甘草甜素(GL)对HepG2.2.15细胞上清HBeAg分泌,Toll样受体4(TLR4)信号分子的表达及对细胞增殖的影响。方法实时荧光定量PCR检测TLR4表达;直接免疫荧光流式细胞术(FCM)检测表达TLR4的阳性细胞率;ELASA检测HBV所分泌抗原;MTT检测细胞增殖活性,并与空白对照组比较。结果给药后HBVe抗原(HBeAg)的分泌结果显示,总体均数间差异显著(P<0.05),但甘草甜素各组与对照组比较差异无统计学意义;GL各组TLR4 mRNA及流式细胞TLR4的表达总体均数间均有显著差异(P<0.01),分别与对照组相比差异有显著性(P<0.01),100μg/mL组尤其明显;MTT实验显示,200μg/mL以下三个剂量组均可促进细胞增殖,50μg/mL组与对照组间差异显著(P<0.05);400μg/mL、800μg/mL两组均显著抑制细胞增殖(P<0.01);而MTT结果与HBeAg水平呈显著负相关(P<0.01),与TLR4表达无相关关系(P>0.05)。结论研究表明,甘草甜素对HepG2.2.15的e抗原分泌可能具有双向作用;细胞的存活率与e抗原呈负相关;TLR4在HepG2.2.15细胞株低表达,GL可上调TLR4表达,甘草甜素可影响先天免疫中的TLR4信号分子,有可能在体内通过免疫途径影响HBV复制和抗原分泌。

慢病毒介导的shRNA干扰人HepG2.2.15细胞Akt2基因表达的研究

目的针对Akt2基因构建shRNA慢病毒干扰载体并评价慢病毒介导的RNA干扰在人HepG2.2.15中的基因沉默效应。方法设计Akt2的RNAi寡聚核苷酸序列,利用慢病毒载体构建Akt2的shRNA载体,转染入大肠埃希菌并观察重组表达状况,利用293T细胞包装得到重组腺病毒,以绿色荧光蛋白(GFP)作为标记,逐孔稀释法确定转染效率及滴度,以实时荧光定量法比较各组对Akt2 mRNA的干扰效果。结果筛选了所构建的3个Akt2靶向序列,包装shRNA慢病毒后转染HepG2.2.15细胞,慢病毒转染后的沉默效率可达85%,比较得出沉默效率最高的靶序列和工作条件。结论本研究成功构建并筛选了针对Akt2的shRNA慢病毒载体,有效抑制HepG2.2.15细胞中Akt2 mRNA的表达。