丙型肝炎病毒核心蛋白稳定表达肝癌细胞株的构建及生物学研究

旨在构建稳定表达HCV核心蛋白的稳定细胞系Huh7-Core并进行初步的生物学功能研究.利用PCR技术扩增HCV核心蛋白基因,通过酶切连接反应将目的基因克隆至载体pSEB-3Flag中,将重组质粒pSEB-3F-Core和辅助质粒pAmpho共转染Huh7细胞,经过Blasticidine抗性筛选,建立稳定表达HCV核心蛋白的肝癌细胞系Huh7-Core.采用RT-PCR、Western blot鉴定Huh7-Core细胞株中核心蛋白的稳定表达并采用MTS、结晶紫试验观察Huh7-Core稳定细胞株的增殖情况.结果显示,成功构建了表达HCV核心蛋白的稳定细胞株Huh7-Core.结晶紫、MTS试验证实Huh7-Core细胞较Huh7-3Flag细胞增殖速度增快,表达HCV核心蛋白的Huh7-Core稳定细胞株构建成功,Core稳定表达后可促进Huh7细胞生长速度.     

Emodin inhibits the growth of hepatoma cells: Finding the common anti-cancer pathway using Huh7, Hep3B, and HepG2 cells

Emodin—a major component of Rheum palmatum L.—exerts antiproliferative effects in cancer cells that are regulated by different signaling pathways. Hepatocellular carcinoma has high-incidence rates and is associated with poor prognosis and high mortality rates. This study was designed to evaluate the effects of emodin on human hepatocarcinoma cell viability and investigate its mechanisms of action in Huh7, Hep3B, and HepG2 cells. To define the molecular changes associated with this process, expression profiles were compared in emodin-treated hepatoma cells by cDNA microarray hybridization, quantitative RT-PCRs, and Western blot analysis. G2/M phase arrest was observed in all 3 cell lines. Cell cycle regulatory gene analysis showed increased protein levels of cyclin A, cyclin B, Chk2, Cdk2, and P27 in hepatoma cells after time courses of emodin treatment, and Western blot analysis showed decreased protein levels of Cdc25c and P21. Microarray expression profile data and quantitative PCR revealed that 15 representative genes were associated with emodin treatment response in hepatoma cell lines. The RNA expression levels of CYP1A1, CYP1B1, GDF15, SERPINE1, SOS1, RASD1, and MRAS were upregulated and those of NR1H4, PALMD, and TXNIP were downregulated in all three hepatoma cells. Moreover, at 6 h after emodin treatment, the levels of GDF15, CYP1A1, CYP1B1, and CYR61 were upregulated. Here, we show that emodin treatment caused G2/M arrest in liver cancer cells and increased the expression levels of various genes both in mRNA and protein level. It is likely that these genes act as biomarkers for hepatocellular carcinoma therapy.     

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1α subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1α as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC₅₀ = 5.16 μM). The mechanism of this inhibition did not involve suppression of HIF-1α protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC₅₀ = 4.75 μM). Exposure of Huh7 cells to 10 μΜ kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 μM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.     

Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI₅₀) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p < 0.0001, T-test). At 8 μg/ml (GI₇₀), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p < 0.05, T-test, n = 8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI₅₀ of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p < 0.05, T-test, n = 9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.     

Enhancement of rhubarb extract solubility and bioactivity by 2-hydroxypropyl-β-cyclodextrin

Rhubarb is a traditional Chinese medicinal herb, and the ethanolic extract of rhubarb consists of active anthraquinones, which are hydrophobic and have antiproliferative effects on hepatoma cell lines. To increase the aqueous solubility of rhubarb and study the consequent bioavailability, the ethanolic extract of rhubarb was complexed with 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide that has a hydrophilic outer surface and a hydrophobic central cavity, to form a rhubarb–HP-β-CD complex. This complex was characterized by performing nuclear magnetic resonance spectroscopy, two-dimensional rotating frame spectroscopy and thin layer chromatography to confirm the inclusion of anthraquinones from rhubarb extract in HP-β-CD (weight ratio of rhubarb extract:HP-β-CD = 1:9). We investigated the effects of complexing rhubarb extract with HP-β-CD on the growth of Huh7 and HepG2 cells by performing cytotoxicity analysis, cellular uptake test, and colony formation assay. Our results showed that complexation of rhubarb extract with HP-β-CD increased the aqueous solubility and bioavailability of rhubarb and thus enhanced its effect on hepatoma cells.     

Estradiol enhances cell-associated paraoxonase 1 (PON1) activity in vitro without altering PON1 expression

PON1 is a high density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. In vivo animal and human studies have indicated that estradiol (E2) supplementation enhances serum PON1 activity. In this study, we sought to determine if E2 directly up-regulates cell-associated PON1 activity in vitro and to characterize the mechanism of regulation. In vitro E2 treatment of both the human hepatoma cell line Huh7 and normal rat hepatocytes resulted in a 2- to 3-fold increase in cell-associated PON1 catalytic activity. E2 potently induced PON1 activity with average EC₅₀ values of 15 nM for normal hepatocytes and 68 nM for Huh7. The enhancement of PON1 activity by E2 was blocked by the estrogen receptor (ER) antagonist ICI 182,780 indicating that E2 was acting through the ER. The up-regulation of PON1 activity by E2 did not involve enhancement of PON1 mRNA or protein levels and did not promote secretion of PON1. Thus, E2 can enhance cell-associated PON1 activity in vitro without altering PON1 gene expression or protein level. Our data suggest that E2 may regulate the specific activity and/or stability of cell surface PON1.     

Hepatitis C Virus Core Protein Down-Regulates p21Waf1/Cip1 and Inhibits Curcumin-Induced Apoptosis through MicroRNA-345 Targeting in Human Hepatoma Cells

Background

Hepatitis C virus (HCV) has been reported to regulate cellular microRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma, but HCV core-modulated cellular microRNAs are unknown. The HCV core protein regulates p21^Waf1/Cip1 expression. However, the mechanism of HCV core-associated p21^Waf1/Cip1 regulation remains to be further clarified. Therefore, we attempted to determine whether HCV core-modulated cellular microRNAs play an important role in regulating p21^Waf1/Cip1 expression in human hepatoma cells.

Methods

Cellular microRNA profiling was investigated in core-overexpressing hepatoma cells using TaqMan low density array. Array data were further confirmed by TaqMan real-time qPCR for single microRNA in core-overexpressing and full-length HCV replicon-expressing cells. The target gene of microRNA was examined by reporter assay. The gene expression was determined by real-time qPCR and Western blotting. Apoptosis was examined by annexin V-FITC apoptosis assay. Cell cycle analysis was performed by propidium iodide staining. Cell proliferation was analyzed by MTT assay.

Results

HCV core protein up- or down-regulated some cellular microRNAs in Huh7 cells. HCV core-induced microRNA-345 suppressed p21^Waf1/Cip1 gene expression through targeting its 3′ untranslated region in human hepatoma cells. Moreover, the core protein inhibited curcumin-induced apoptosis through p21^Waf1/Cip1-targeting microRNA-345 in Huh7 cells.

Conclusion and Significance

HCV core protein enhances the expression of microRNA-345 which then down-regulates p21^Waf1/Cip1 expression. It is the first time that HCV core protein has ever been shown to suppress p21^Waf1/Cip1 gene expression through miR-345 targeting.     

Cysteine-rich secretory protein 3 inhibits hepatitis C virus at the initial phase of infection

Hepatitis C virus (HCV) affects 2–3% of the global population. Approximately one-quarter of acute infections cause chronic hepatitis that leads to liver cirrhosis or hepatocellular carcinoma. The major obstacle of current research is the extremely narrow host tropism of HCV. A single HCV strain can replicate in the Huh7 human hepatoma cell line. Huh7 cells can be adapted under selective pressure in vitro to identify host factors that influence viral replication. Here, we extended this strategy to the in vivo condition and generated a series of cell lines by multiple rounds of adaptation in immunocompromised mice. Adaptation increased the cellular resistance to HCV infection. Microarray analyses revealed that the expression levels of several genes were associated with HCV resistance. Notably, up-regulation of the mRNA encoding cysteine-rich secretory protein 3 (CRISP3), a glycoprotein with unknown function that is secreted from multiple exocrine glands, was correlated with HCV resistance. The presence of CRISP3 in the culture medium limited HCV replication at the early phase of infection.     

氢分子对肝癌细胞Huh7的影响

氢分子对癌症等疾病具有良好的治疗或改善效果,并且获得简单、使用简便且无副作用。为了明确氢分子对肝癌细胞Huh7的影响,从细胞活性、细胞周期和凋亡、细胞成瘤能力、细胞迁移侵袭等方面研究了氢分子对肝癌细胞Huh7的作用效果。细胞活性检测(CCK-8法)结果表明,氢分子对Huh7细胞活性具有明显的抑制效果。细胞成瘤能力实验中,克隆球实验显示,含氢培养基组(H)细胞成球数目有所减少,同时克隆球直径大小显著降低(P<0.0001);平板克隆实验显示,H组细胞克隆的数量也显著减少(P<0.001),说明氢分子可以抑制Huh7细胞的干性。氢分子对Huh7细胞周期的影响也较为明显,且具有促凋亡的作用和抑制细胞迁移与浸润的效果。同时,氢分子对细胞内中间丝波形蛋白的表达也具有抑制作用。研究表明,氢分子降低了Huh7细胞的活性和增殖能力,减弱了Huh7细胞的干性,同时抑制了细胞侵袭及迁移的能力,降低了细胞波形蛋白的表达,为氢分子用于肝癌防治提供了一定的实验基础。     

Enhancement of expression of survivin promoter-driven CD/TK double suicide genes by the nuclear matrix attachment region in transgenic gastric cancer cells

This work aimed to study a novel transgenic expression system of the CD/TK double suicide genes enhanced by the nuclear matrix attachment region (MAR) for gene therapy. The recombinant vector pMS-CD/TK containing the MAR–survivin promoter–CD/TK cassette was developed and transfected into human gastric cancer SGC-7901 cells. Expression of the CD/TK genes was detected by quantitative real-time PCR (qPCR) and Western blot. Cell viability and apoptosis were measured using the methyl thiazolyl tetrazolium (MTT) assay and flow cytometry. When the MAR fragment was inserted into the upstream of the survivin promoter, the qPCR result showed that the expression of the CD/TK genes significantly increased 7.7-fold in the transgenic SGC-7901 cells with plasmid pMS-CD/TK compared with that without MAR. MTT and flow cytometry analyses indicated that treatment with the prodrugs (5-FC + GCV) significantly decreased the cellular survival rate and enhanced the cellular apoptosis in the SGC-7901 cells. The expression of the CD/TK double suicide genes driven by the survivin promoter can be enhanced by the MAR fragment in human gastric cancer cells.     

Smo基因在人肝癌Huh-7细胞中的表达及小RNA干扰Smo基因表达对肝癌Huh-7细胞增殖及凋亡的影响

目的:在细胞学层面上研究Smo基因在人肝癌Huh-7细胞中的表达及小RNA干扰Smo基因表达对肝癌Huh-7细胞增殖及凋亡的影响。方法:Huh-7细胞培养,总RNA抽提,紫外分光光度计纯度测定,Western印记法检测Smo蛋白表达,转染后流式细胞检测Huh-7凋亡率。结果:在mRNA和蛋白水平Smo均强表达。siRNA-1干扰序列干扰结果最强,转染后可诱导Huh-7细胞凋亡。结论:siRNA-l能对肝癌Huh7细胞Smo基因表达产生干涉作用,siRNA-1序列能有效地降解肝癌Huh7细胞内的SmomRNA,使Smo mRNA及Smo蛋白表达下调,从而达到沉默肝癌Huh7细胞中Smo mRNA表达的效果。     

Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9

Expression of MACC1 (metastasis-associated in colon cancer-1) protein is associated with metastasis of various human cancers. This study analyzed MACC1 protein expression in hepatocellular carcinoma (HCC) tissue specimens and then investigated the effects of MACC1 knockdown on HCC cell migration and invasion, and gene expression levels. Sixty pairs of HCC and adjacent normal liver tissues from HCC patients were analyzed for MACC1 expression immunohistochemically. The HCC cell lines Hep3B, Huh7, MHCC97H, SMMC-7721, Bel-7402, and HepG2 and the normal liver cell line LO2 were used to assess expressions of MACC1 mRNA and MACC1 protein using qRT-PCR and western blot, respectively. MACC1 short hairpin RNA (shRNA) was used to knockdown MACC1 protein expression in Huh7 cells. Changes in the tumor phenotype of these cells were analyzed with wound healing assay and invasion assays, and differences in gene expression were evaluated via western blot. Immunofluorescence was used to locate MACC1 protein in the above cell lines. MACC1 was highly expressed in HCC tissues and the nuclear expression of MACC1 protein was associated with poor tumor differentiation and intrahepatic metastasis or portal invasion. Moreover, MACC1 mRNA and MACC1 protein was also expressed in HCC cell lines. Immunostaining showed that MACC1 protein was localized in both nuclei and cytoplasm of HCC cell lines and the nuclear localization of MACC1 protein was associated with increased aggressiveness of HCC in cell lines. Knockdown of MACC1 expression using MACC1-shRNA reduced Huh7 cell migration and invasion abilities, which was associated with downregulation of MMP2, MMP9, and c-Met proteins in Huh7 cells. Localization of MACC1 protein to the nucleus may predict HCC progression. Knockdown of MACC1 expression using MACC1 shRNA warrants further evaluation as a novel therapeutic strategy for control of HCC.     

Characterization of infectious hepatitis C virus from liver-derived cell lines

The efficient production of infectious HCV from the JFH-1 strain is restricted to the Huh7 cell line and its derivatives. However, the factors involved in this restriction are unknown. In this study, we examined the production of infectious HCV from other liver-derived cell lines, and characterized the produced viruses. Clones of the Huh7, HepG2, and IMY-N9, harboring the JFH-1 full-genomic replicon, were obtained. The supernatant of each cell clone exhibited infectivity for naïve Huh7. Each infectious supernatant was then characterized by sucrose density gradient. For all of the cell lines, the main peak of the HCV-core protein and RNA exhibited at approximately 1.15 g/mL of buoyant density. However, the supernatant from the IMY-N9 differed from that of Huh7 in the ratio of core:RNA at 1.15 g/mL and significant peaks were also observed at lower density. The virus particles produced from the different cell lines may have different characteristics.     

GALNT11 as a new molecular marker in chronic lymphocytic leukemia

Aberrant mucin O-glycosylation often occurs in different cancers and is characterized by immature expression of simple mucin-type carbohydrates. At present, there are some controversial reports about the Tn antigen (GalNAcα-O-Ser/Thr) expression and there is a great lack of information about the [UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-Ts)] expression in chronic lymphocytic leukemia (CLL). To gain insight in these issues we evaluated the Tn antigen expression in CLL patient samples using two Tn binding proteins with different fine specificity. We also studied the expression from 14 GalNAc-Ts genes in CLL patients by RT-PCR. Our results have provided additional information about the expression level of the Tn antigen, suggesting that a low density of Tn residues is expressed in CLL cells. We also found that GALNT11 was expressed in CLL cells and normal T cell whereas little or no expression was found in normal B cells. Based on these results, GALNT11 expression was assessed by qPCR in a cohort of 50 CLL patients. We found significant over-expression of GALNT11 in 96% of B–CLL cells when compared to normal B cells. Moreover, we confirmed the expression of this enzyme at the protein level. Finally we found that GALNT11 expression was significantly associated with the mutational status of the immunoglobulin heavy chain variable region (IGHV), [?²(1) = 18.26; P < 0.0001], lipoprotein lipase expression [?²(1) = 13.72; P = 0.0002] and disease prognosis [?²(1) = 15.49; P < 0.0001]. Our evidence suggests that CLL patient samples harbor aberrant O-glycosylation highlighted by Tn antigen expression and that the over-expression of GALNT11 constitutes a new molecular marker for CLL.     

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV

Type I and III interferons induce expression of the “myxovirus resistance proteins” MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles (“biomolecular condensates”). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40–50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20–30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.     

肝细胞癌组织中OATP1B3的表达及其作用的初步研究

摘要 目的：探讨有机阴离子转运多肽1B3（OATP1B3）在肝细胞癌（肝癌）组织中的表达及作用。方法：通过免疫组化实验和免疫印迹试验（Western blot）检测肝癌组织及其癌旁组织中OATP1B3情况,并分析其与患者临床病理特征的相关性。采用实时荧光定量聚合酶链反应（qRT-PCR）检测OATP1B3在多株肝癌细胞中的表达情况,选择表达量相对较低的人肝癌细胞（HepG2和Huh7）细胞进行过表达实验,细胞毒实验（MTT）和流式细胞术分别检测OATP1B3对细胞增殖及凋亡的影响。结果：肝癌组织中OATP1B3表达水平明显低于癌旁组织（P<0.05）,且与患者恶性肿瘤国际临床病期分类（TNM分期）、肿瘤分化程度、有无肿瘤复发显著相关（P<0.05）。过表达OATP1B3可抑制HepG2和Huh7细胞增殖,促进细胞凋亡。结论：OATP1B3在肝癌组织中低表达,上调其表达可抑制肝癌细胞增殖和促进细胞凋亡。OATP1B3可能是肝癌的抑癌基因,对肝癌的发生、发展具有重要作用。     

Luteolin and Apigenin Attenuate 4-Hydroxy-2-Nonenal-Mediated Cell Death through Modulation of UPR,Nrf2-ARE and MAPK Pathways in PC12 Cells

Luteolin and apigenin are dietary flavones and exhibit a broad spectrum of biological activities including antioxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) has been implicated as a causative agent in the development of neurodegenerative disorders. This study investigates the cytoprotective effects of luteolin and apigenin against 4-HNE-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Both flavones restored cell viability and repressed caspase-3 and PARP-1 activation in 4-HNE-treated cells. Luteolin also mitigated 4-HNE-mediated LC3 conversion and reactive oxygen species (ROS) production. Luteolin and apigenin up-regulated 4-HNE-mediated unfolded protein response (UPR), leading to an increase in endoplasmic reticulum chaperone GRP78 and decrease in the expression of UPR-targeted pro-apoptotic genes. They also induced the expression of Nrf2-targeted HO-1 and xCT in the absence of 4-HNE, but counteracted their expression in the presence of 4-HNE. Moreover, we found that JNK and p38 MAPK inhibitors significantly antagonized the increase in cell viability induced by luteolin and apigenin. Consistently, enhanced phosphorylation of JNK and p38 MAPK was observed in luteolin- and apigenin-treated cells. In conclusion, this result shows that luteolin and apigenin activate MAPK and Nrf2 signaling, which elicit adaptive cellular stress response pathways, restore 4-HNE-induced ER homeostasis and inhibit cytotoxicity. Luteolin exerts a stronger cytoprotective effect than apigenin possibly due to its higher MAPK, Nrf2 and UPR activation, and ROS scavenging activity.     

Lipopolysaccharide increases gastric and circulating NUCB2/nesfatin-1 concentrations in rats

Bacterial lipopolysaccharide (LPS) is an established animal model to study the innate immune response to Gram-negative bacteria mimicking symptoms of infection including reduction of food intake. LPS decreases acyl ghrelin associated with decreased concentrations of circulating ghrelin-O-acyltransferase (GOAT) likely contributing to the anorexigenic effect. We also recently described the prominent expression of the novel anorexigenic hormone, nucleobindin2 (NUCB2)/nesfatin-1 in gastric X/A-like cells co-localized with ghrelin in different pools of vesicles. To investigate whether LPS would affect gastric and circulating NUCB2/nesfatin-1 concentration, ad libitum fed rats were equipped with an intravenous (iv) catheter. LPS was injected intraperitoneally (ip, 100 μg/kg) and blood was withdrawn before and at 2, 5, 7 and 24 h post injection and processed for NUCB2/nesfatin-1 radioimmunoassay. Gastric corpus was collected to measure NUCB2 mRNA expression by RT-qPCR and NUCB2/nesfatin-1 protein concentration by Western blot. Injection of LPS increased plasma NUCB2/nesfatin-1 concentrations by 43%, 78% and 62% compared to vehicle at 2 h, 5 h and 7 h post injection respectively (p < 0.05) and returned to baseline at 24 h. The plasma NUCB2/nesfatin-1 increase at 2 h was associated with increased corpus NUCB2 mRNA expression (p < 0.01), whereas NUCB2 mRNA was not detectable in white blood cells. Likewise, gastric NUCB2 protein concentration was increased by 62% after LPS compared to vehicle (p < 0.01). These data show that gastric NUCB2 production and release are increased in response to LPS. These changes are opposite to those of ghrelin in response to LPS supporting a differential gastric regulation of NUCB2/nesfatin-1 and ghrelin expression derived from the same cell by immune challenge.     

丙型肝炎病毒高水平复制细胞株的构建及其机制初步研究

旨在探讨丙型肝炎病毒(hepatitis C virus, HCV)cured细胞株的易感机制。本研究将体外转录的HCV RNA电转入肝癌细胞系Huh 7细胞,建立HCV复制子细胞株,用 γ-干扰素(interferon,IFN)处理复制子细胞株,获得HCV cured Huh 7A和Huh 7B细胞株。用插入报告基因的HCV毒株Jc1-G感染上述细胞株,分别进行荧光素酶活性测定、蛋白质印迹法和荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测以验证其易感性。收集Huh 7、Huh 7.5、Huh 7A和Huh 7B细胞并利用IFN-α处理,之后用蛋白质印迹法及荧光定量PCR进行检测,验证细胞株中IFN诱生信号通路中关键因子内源性表达及抗病毒活性ISGs的激活水平。结果显示,在Huh 7A和Huh 7B细胞中检测不到病毒RNA,与Huh 7细胞一致。病毒感染实验中,与Huh 7细胞相比,Huh 7A和Huh 7B细胞株中荧光素酶活性增高百倍,病毒蛋白表达和RNA水平亦显著上调,与Huh 7.5细胞株中的表达水平接近。IFN信号通路实验中,与Huh 7细胞相比,Huh 7A和Huh 7B细胞株中RIG-I/MDA5/MAVS内源性蛋白表达和mRNA水平无明显差异;IFN-α处理细胞后IFN刺激基因isg56,mx1,mx2,oax1,oax2,viperin,cxcl10,ifitm1和ifitm3激活水平亦无显著变化。结果提示,本研究制备的Huh 7A和Huh 7B细胞株可支持HCV高水平复制,将为研究病毒复制机制提供有力的支持。     

Stromal cell-derived factor-1/chemokine (C-X-C motif) ligand 12 stimulates human hepatoma cell growth, migration, and invasion

In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The aim of this study was to determine whether the chemokine stromal cell-derived factor 1 (SDF-1) induces the growth, migration, and invasion of human hepatoma cells. We show that SDF-1 G protein-coupled receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), and SDF-1 mRNA are expressed in human hepatoma Huh7 cells, which secrete and bind SDF-1. This binding depends on CXCR4 and glycosaminoglycans. SDF-1 associates with CXCR4, and syndecan-4 (SDC-4), a heparan sulfate proteoglycan at the plasma membrane of Huh7 cells, induces the growth of Huh7 cells by promoting their entry into the cell cycle, and inhibits the tumor necrosis factor-alpha-mediated apoptosis of the cells. SDF-1 also reorganizes Huh7 cytoskeleton and induces tyrosine phosphorylation of focal adhesion kinase. Finally, SDF-1 activates matrix metalloproteinase-9, resulting in increased migration and invasion of Huh7 cells. These biological effects of SDF-1 were strongly inhibited by the CXCR4 antagonist AMD3100, by a glycosaminoglycan, heparin, as well as by beta-D-xyloside treatment of the cells, or by c-jun NH(2)-terminal kinase/stress-activated protein kinase inhibitor. Therefore, the CXCR4, glycosaminoglycans, and the mitogen-activated protein kinase signaling pathways are involved in these events. The fact that reducing SDC-4 expression by RNA interference decreased SDF-1-induced Huh7 hepatoma cell migration and invasion strongly indicates that SDC-4 may be an auxiliary receptor for SDF-1. Finally, the fact that CXCR4 is expressed in hepatocellular carcinoma cells from liver biopsies indicates that the in vitro results reported here could be extended to in vivo conditions.