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.

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.

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.

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。

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。

HBIG-PBCA-NP对QSG、HepG及PBMC细胞增长率及毒性的影响

目的研究乙型肝炎免疫球蛋白（HBIG）聚氰基丙烯酸正丁酯纳米粒（HBIG—PBCA—NP）、PBCA—NP的体外应用安全性。方法用乳化聚合法分别制备HBIG-PBCA-NP、PBCA-NP；采用MTT比色法研究不同浓度的HBIG-PBCA-NP、PBCA—NP对外周血单核细胞（PBMC）、QSG7701细胞及HepG2.2．15细胞生长抑制作用的影响；用自动生化仪测定乳酸脱氢酶（LDH）、丙氨酸转氨酶（ALT）活性。结果按相对生长率（RGR％），HBIG—PBCA—NP、PBCA—NP对PBMC、QSG7701细胞及HepG2．2．15细胞的毒性分级分别为0—1级，LDH与ALT释放值与HBIG、空白对照相比，差异无显著性（P〉0．01）。结论PBCA—NP属无毒级载体，HBIG—PBCA—NP具有良好的细胞生物安全性。

Effects of SAHA on proliferation and apoptosis of hepatocellular carcinoma cells and hepatitis B virus replication

AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.

琐琐葡萄多糖体外抗乙型肝炎病毒作用的实验研究

琐琐葡萄（Vitis viniferal L）为葡萄科小无核红葡萄,药用果实,主产于我国新疆吐鲁番、和田、鄯善等地[1],具有健脾胃、理肺、生津、养血等功效;在维吾尔医临床主要应用于脾胃不和、神志不安以及小儿麻疹和肝炎等病症的治疗,收载于《神农本草经》、《本草纲目》和《维吾尔药志》等历代医药文献中。

裙带菜多糖抗肿瘤作用的研究

[目的]观察裙带菜多糖的体内、外抗肿瘤作用,并对其机制进行讨论。[方法]采用MTT法观察裙带菜多糖体外对人肝癌细胞HepG-2的抑制作用;通过对615荷瘤小鼠瘤重及免疫器官脾脏的研究,观察了裙带菜多糖的体内抗肿瘤作用。[结果]与阴性对照组相比裙带菜多糖各浓度组体外对人肝癌细胞HepG-2均有很强的抑制作用(P<0.01),其中15、10 mg/mL组的抑制率分别为57.20%、51.79%,略低于5-FU组;在荷瘤小鼠实验中,与空白对照组相比裙带菜多糖各浓度组均有很强的肿瘤抑制作用,其中400、200 mg/kg组抑瘤率分别为69.13%、42.11%,与5-FU组相近。[结论]裙带菜多糖对人肝癌细胞HepG-2抑制效果显著,小鼠体内实验也证实了裙带菜多糖较强的抗肿瘤作用。

毛鸡骨草醇提液对HepG2.2.15细胞乙型肝炎表面抗原及乙型肝炎E抗原的影响

目的:观察毛鸡骨草体外抗乙型肝炎病毒(HBV)的作用。方法:采用MTT法检测毛鸡骨草对HepG2.2.15细胞的半数毒性浓度(TC50)和最大无毒浓度(TC0),采用酶联免疫吸附实验(ELISA)法测定<TC0的5个不同浓度的毛鸡骨草醇提液对HepG2.2.15细胞株表达的乙型肝炎表面抗原(HBsAg)和乙型肝炎E抗原(HBeAg)的影响。结果:毛鸡骨草醇提取液可有效地抑制细胞HBsAg和HBeAg的分泌;在质量浓度为4 g.L-1作用144 h对HBsAg,HBeAg抑制作用最为明显,抑制率分别为33.1%,40.1%。结论:毛鸡骨草在体外有一定的抗HBV作用。

太白楤木总皂苷对HBV DNA的影响

目的:观察太白楤木总皂苷在体外对乙型肝炎病毒脱氧核糖核酸(HBV DNA)的影响。方法:以HepG2.2.15细胞为靶细胞,分别用不同浓度的太白楤木总皂苷和拉米夫定培养液培养,采用荧光定量PCR测定细胞外HBV DNA含量,评价太白楤木总皂苷对体外HBV DNA的抑制作用。结果:太白楤木总皂苷对体外培养HepG 2.2.15细胞分泌的HBV DNA 50%抑制浓度(IC50)为0.128g/L,治疗指数(TI)为5.11g/L。结论:太白楤木总皂苷具有一定的体外抗HBV作用。

甲基斑蝥胺抗乙型肝炎病毒体外实验研究

用２．２．１５细胞株体外抗ＨＢＶ药物模型，对研制的甲基斑蝥胺药物体外抗ＨＢＶ流行性进行评价。应用固相放射免疫测定法检测药物对２．２．１５细胞所分泌的ＨＢｓＡｇ、ＨＢｅＡｇ的抑制效果，应用Ｓｏｕｔｈｅｒｎ转膜杂交法分析药物对ＨＢＶ－ＤＮＡ的区带影响，同时以ＭＴＴ法检测药物细胞毒性。药物对病毒复制指标ＨＢｓＡｇ、ＨＢｅＡｇ的５０％抑制浓度（ＩＤ５０）分别为２．８３ｇ／Ｌ、４．６０ｇ／Ｌ，该药对ＨＢＶ－ＤＮＡ区带无明显影响，药物对细胞的５０％毒性浓度（ＣＤ５０）１０．６０ｇ／Ｌ。药物对ＨＢｓＡｇ、ＨＢｅＡｇ的治疗指数（ＴＩ）＝ＣＤ５０／ＩＤ５０分别为３．７５、２．３０。说明甲基斑蝥胺药物在体外具有一定抗ＨＢＶ活性。

补肾冲剂体外抗乙型肝炎病毒作用研究

目的:研究中药复方补肾冲剂体外抗HBV活性。方法:采用血清药理学方法,在HBV的体外细胞培养系统中(2.2.15细胞)进行补肾冲剂抗HBV作用观察,含药鸭血清中的细胞培养上清中HBsAg/HBeAg检测采用ELISA法,MTT法测定细胞存活率,并计算ID50、CD50和TI。结果:补肾冲剂在最佳鸭血清添加量(40L)的浓度下,当鸭血清添加量分别为20%、40%、60%、80%时对HBsAg分泌的抑制率分别是19%、40%、68%、89%,治疗指数为2.31,对HBeAg分泌的抑制率分别是17%、28%、48%、70%,治疗指数为2.45。结论:补肾冲剂可能有一定的体外抗HBV活性。

黄芪抗乙型肝炎病毒的体外实验研究

目的探讨黄芪对乙型肝炎病毒抗原表达的抑制作用。方法黄芪作用于体外培养的HepG2.2.15细胞,观察黄芪对HepG2.2.15细胞分泌HBsAg,HBeAg的影响及药物的细胞毒性。结果黄芪作用于HepG2.2.15细胞12d后,对细胞的半数毒性浓度(CD50)1500ng/ml,对HBsAg和HBeAg抑制的半数有效浓度(ID50)分别为115.4μg/ml和317.2μg/ml,黄芪对HBsAg和HBeAg的治疗指数(TI)分别13.00,4.73。结论黄芪在体外细胞培养中对HBsAg及HBeAg的分泌有较好的抑制作用。

苎麻绿原酸体外抗乙型肝炎病毒的作用研究

为了考察苎麻绿原酸对HepG 2 2.2.15细胞毒性作用,以及对其HBsAg、HBeAg表达的抑制作用,本研究提取苎麻中的绿原酸,配成100.00μg/mL、50.00μg/mL、25.00μg/mL、12.50μg/mL和6.25μg/mL 5个浓度,作用于HepG 2 2.2.15细胞,在第3日、第6日和第9日分别收集上清液,四甲基噻唑蓝(MTT)法检测药物的细胞毒性;酶联免疫(ELISA)法检测HBsAg和HBeAg水平。实验结果表明:苎麻绿原酸对HepG 2 2.2.15细胞无毒性作用,其对HBsAg的表达呈现一定的抑制作用,且呈时间依赖性和浓度依赖性抑制,在第9日,最高浓度100.00μg/mL时抑制率为70.12%;对HBeAg的分泌无抑制作用。苎麻绿原酸无细胞毒性作用,能明显抑制HepG 2 2.2.15细胞HBsAg的表达。

肝必康胶囊体外抗乙肝病毒的实验研究

目的:观察肝必康胶囊对乙肝病毒的抑制作用。方法:采用HepG 2.2.15细胞的培养,MTT比色法检测药物对细胞的毒性,定量PCR检测HBV-DNA,ELISA法检测HBsAg、HBeAg的含量,研究肝必康胶囊对HepG 2.2.15细胞的毒性,HBsAg与HBeAg表达及HBV-DNA含量的影响。结果:肝必康半数细胞毒浓度(TC50)为4.677 mg/mL,最大无毒浓度(TC0)为0.118 mg/mL。各组在用药后72、144 h细胞上清中HBV DNA拷贝数降低,肝必康在0.1 mg/mL时抑制率最高。各组用药后细胞培养上清液中的HBsAg和HBeAg浓度显著降低。肝必康在0.1 mg/mL时抑制率最高。肝必康各组含药血清在用药后72、144 h细胞上清中HBsAg和HBeAg浓度显著降低,高剂量组抑制率最高。显示明显抗HBV的作用,TI均大于2。结论:肝必康胶囊体外培养细胞实验证实其有较强的抗乙肝病毒作用,且毒性较低。

知母宁抗乙肝病毒作用的体内外实验研究

目的:观察知母宁的抗乙肝病毒作用。方法:观察不同浓度知母宁对体外培养的2.2.15细胞分泌HBsAg、HBeAg的抑制作用及对乙肝模型鸭DHBV-DNA的影响。结果:知母宁浓度为4mg/mL时对2.2.15细胞分泌HBeAg有明显的抑制作用;以200mg/kg治疗乙肝模型鸭10d后DHBV-DNA水平明显降低。结论:知母宁有抗乙肝病毒的作用,其机制可能与抑制HBV-DNA的复制有关。