大豆高支低芳氨基酸混合物在肝功不全治疗中的应用

大豆高支低芳氨基酸混合物是从大豆蛋白质中制取的一种特殊组成的氨基酸混合物。其支链氨基酸含量占51％,支链氨基酸/芳香族氨基酸克分子比(简称支/芳比)达51.8。动物实验表明,该种氨基酸混合物能提高实验性肝性脑病大鼠的血清支/芳比,纠正氨基酸谱紊乱,增加动物活存数,减轻肝性脑病症状。临床应用结果显示,用该种氨基酸混合物配制的肝氨要素,也能提高肝功不全患者的血清支/芳比,纠正血清氨基酸谱紊乱,提高血清白蛋白含量,促进肝腹水消退,改善肝性脑病的症状。这些结果提示,大豆高支低芳氨基酸混合物对肝功不全的治疗具有显著作用。     

米非司酮对子宫内膜癌细胞增殖抑制的研究

目的:探讨孕激素拮抗剂米非司酮对患者子宫内膜癌细胞凋亡、周期及孕激素受体(PR)亚型的影响。方法:患者在米非司酮治疗前及治疗后分别留取标本60例,用流式细胞仪检测组织细胞凋亡率和周期时相的变化,用实时定量PCR检测PR-A mRNA和PR-B mRNA的表达情况。结果:患者经过米非司酮治疗后,癌细胞凋亡率和G0/G1比率升高,S期比率下降,RR-B mRNA表达显著下降,PR-B与PR-A的比例明显降低。结论:米非司酮下调PR-B的表达,使癌细胞阻滞于G1期,促进癌细胞凋亡,抑制子宫内膜癌肿瘤细胞的生长。     

肝癌细胞中抑制Galectin-3基因表达对细胞增殖及侵袭能力的影响及机制研究

目的 探讨Galectin-3基因在肝癌细胞中的表达及抑制其表达对肝癌细胞增殖和侵袭能力的影响及机制。 方法 RT-PCR检测人肝癌细胞MHCC-97H、HepG2、SMCC-7721及人正常肝细胞HL-7702中Galectin-3基因的mRNA表达;Control、NC-siRNA、Galectin-3-siRNA转染HepG2细胞,48 h后Western blot检测各组细胞中Galectin-3、MMP-2、MMP-9、Notch1、Hes1的蛋白表达;CCK8实验和流式细胞仪分别检测细胞的增殖和侵袭能力。 结果 MHCC-97H、HepG2、SMCC-7721细胞中Galectin-3的mRNA表达均显著高于正常肝细胞HL-7702中的Galectin-3的mRNA表达,差异有统计学意义(P<0.01),Galectin-3在HepG2细胞中的表达最高,选择作为后续的研究对象;转染siRNA后能显著抑制Galectin-3基因的表达;Galectin-3-siRNA组细胞存活率、细胞侵袭数及MMP-2、MMP-9、Notch1、Hes1蛋白表达均低于对照组和NC-siRNA组,差异有统计学意义(P<0.01)。 结论 抑制肝癌细胞中Galectin-3基因表达能显著降低癌细胞的增殖及侵袭能力,其机制与Notch1信号通路的调控有关。     

GP73表达调控的肝癌细胞模型的筛选和构建

目的 筛选合适的细胞株构建干扰和过表达GP73的肝癌细胞模型,为进一步深入研究GP73调控肝癌的机制提供合适的细胞模型.方法 挑选8株肝癌细胞:Huh-7、Hep3B、MHCC-LM3、MHCC97-L、MHCC97-H、SNU-449、SNU-398、SNU-182,采用qPCR和Western Blot验证GP73...     

Tetrahydro Iso-Alpha Acids and Hexahydro Iso-Alpha Acids from Hops Inhibit Proliferation of Human Hepatocarcinoma Cell Lines and Reduce Diethylnitrosamine Induced Liver Tumor Formation in Rats

Chronic inflammation plays important role in the pathogenesis of hepatocellular carcinoma (HCC). To date, no antiinflammatory approach has shown its efficacy in preventing HCC occurrence in humans. Because tetra- and hexahydro isoalpha acids (THIAA and HHIAA) from hops elicit antiinflammatory properties, we evaluated these compounds for antitumor effects in vitro in human HCC cell lines (HepG2, Hep3B, Huh7) and in vivo in diethylnitrosamine (DEN)-induced animal model of HCC. In human HCC cell lines, THIAA and HHIAA reduced cell proliferation and viability which was associated with the inhibition of the NF-κB-DNA binding and tumor necrosis factor α mRNA expression. Both compounds also inhibited phosphorylation of the mTOR effector p70S6 kinase without affecting ERK, AKT, JNK, and GSK3β phosphorylation or activator protein-1 activation. In DEN-treated rats, administration of THIAA and HHIAA in food reduced the tumor numbers and the expression of the cellular transformation marker glutathione-S-transferase in the liver. In conclusion, THIAA and HHIAA show antitumor properties in vitro in human HCC cell lines as well as in vivo in a chemically induced animal model of HCC.     

苯并（a）芘调节脂代谢促进Hep G2细胞脂质沉积

目的 探索苯并(a)芘（BaP）对人肝癌细胞株Hep G2细胞脂质含量及脂代谢的影响。方法 0、0.01、1nmol/LBaP作用Hep G2细胞，采用刃天青检测细胞活性，油红O染色、甘油三脂试剂盒检测细胞内甘油三脂含量，实时荧光定量PCR检测脂代谢相关调节因子的mRNA表达。结果 经BaP处理后，Hep G2细胞活性不变，细胞内脂质增加，LXR-α、FANS、MPC1、MPC2、CD36的mRNA表达增加，DAGT1、MPT的mRNA表达下降（P＜0.05），FABP1的mRNA表达没有明显改变。结论 BaP影响Hep G2细胞脂质代谢，促进细胞脂质沉积。     

The role of interleukin-1 beta in the regulation of liver-specific gene expression in human hepatoblastoma cells

The authors studied the function of liver cells--on level of DNA and gene regulation--by methods of molecular biology. They found that treating human hepatoma (Hep G2) cells with interleukin-1 (IL-1) leads to the induction of alpha-1-acidglycoprotein (AGP) and complement 3 (C3) mRNA synthesis, and to a concomitant downregulation of albumin (alb), alpha-fetoprotein (AFP) and alpha-2-macroglobulin (alpha 2M) mRNA synthesis. Levels of specific mRNA were measured by Northern-blot analysis. They conclude that Hep G2 cells may serve as a suitable in vitro model for study of the liver specific gene expression, and IL-1 is one of the mediators of these gene control. The regulation is pretranslational as the direction of change in specific mRNA corresponds to the changes in synthesis of the respective proteins.     

Variations in plasma amino acids in septic patients subjected to parenteral nutrition with a high proportion of branched-chain amino acids.

Sepsis is characterized by an increase in the plasma concentration of aromatic amino acids (AAAs) and those containing sulfur and a decrease in the branched-chain amino acids (BCAAs). We studied changes in the plasma aminogram of septic patients given different types of total parenteral nutrition (TPN), analyzing variations in accordance with the type of TPN used and the importance that the use of BCAA may have in these patients. We studied 80 patients with peritonitis divided into two groups of 40 patients each: group 1 was given a solution with 22.5% BCAA and group 2 a solution with 45% BCAA. High BCAA content caused an increase in the plasma concentrations of these amino acids and in the BCAA/AAA quotient and a decrease in AAAs. Plasma concentrations of leucine and valine reached high, potentially toxic levels at 15 days when solutions with high BCAA content were used. Glycine increased in group 1, which may be important because of its tendency to produce hyperammonemia. BCAAs are of unquestioned nutritional importance in view of the evidence of changes that take place in muscle protein catabolism and in plasma amino acids. In the phase of increased protein catabolism, we saw a plasma amino acid pattern in keeping with the existing metabolic situation. The need for BCAA diminishes when the hypercatabolic state disappears.     

Differential inhibition of human cancer cell proliferation by citrus limonoids

Limonoids have been shown to inhibit the growth of estrogen receptor-negative and -positive human breast cancer cells in culture. The primary objective of this study was to test the antiproliferative activity of limonoids (obacunone 17 beta-D-glucopyranoside, nomilinic acid 17 beta-D-glucopyranoside, limonin, nomilin, and a limonoid glucoside mixture), found in high concentrations in mandarin (Citrus reticulata Blanco), against a series of human cancer cell lines. The human cancer cell lines included leukemia (HL-60), ovary (SKOV-3), cervix (HeLa), stomach (NCI-SNU-1), liver (Hep G2), and breast (MCF-7). The growth-inhibitory effects of the four limonoids and the limonoid glucoside mixture against MCF-7 cells were significant, and the antiproliferative activity of the different citrus limonoids was also dose and time dependent. No significant effects were observed on growth of the other cancer cell lines treated with the four individual limonoids at 100 micrograms/ml. At 100 micrograms/ml, the limonoid glucoside mixture demonstrated a partial inhibitory effect on SKOV-3 cancer cells. With use of flow cytometry, it was found that all the limonoid samples could induce apoptosis in MCF-7 cells at relatively high concentrations (100 micrograms/ml). Considering the high concentration needed to induce apoptosis, it is unlikely that this is the primary mechanism of action for the cytotoxic effects seen with limonoids in this study. Further work is needed in this area to establish the mechanism of action of citrus limonoids on human breast cancer cells.     

重组人生长激素联合5-氟尿嘧啶对表达或不表达生长激素受体人肝癌细胞的体外干预

目的研究重组人生长激素（rhGH）在体外对表达或不表达生长激素受体（GHR）的人肝癌细胞系的影响。方法采用免疫组织化学方法检测人肝癌细胞系的GHR表达。每种肿瘤细胞系分4组进行处理：未处理组、5-氟尿嘧啶（5-Fu）处理组、rhGH处理组及5-Fu＋rhGH联合处理组。采用四甲基偶氮唑蓝（MTT）比色法及流式细胞术分析不同浓度rhGH及其与5-Fu合用对人肝癌细胞系的生长抑制、凋亡、细胞周期及增殖指数（PJ）等的影响。结果Bel-7402细胞表达GHR；与未处理组相比，各浓度rhGH组的生长率、G2／M期比例和PI显著升高，细胞凋亡率显著降低（P均〈0．05）；与5-Fu处理组相比，各浓度rhGH＋5-Fu组的G2／M期比例和PI显著升高，抑制率和细胞凋亡率显著降低（P均〈0．05）。SMMC7721不表达GHR；不同浓度rhGH对该细胞系的分裂增殖无明显影响（P均〉0．05）；rhGH＋5-Fu联合处理组与5-Fu处理组问的细胞抑制率、凋亡率及Pl差异也无显著性（P均〉0．05）。结论rhGH在体外能促进GHR^＋的肝癌细胞增殖，减弱5-Fu对GHR^＋细胞的抗癌作用，但对GHR^-的肝癌细胞无明显影响，也不能明显干扰5-Fu对GHR^-细胞的抗癌功能。     

Sodium butyrate inhibits cell growth and stimulates p21WAF1/CIP1 protein in human colonic adenocarcinoma cells independently of p53 status

Butyric acid, one of the short-chain fatty acids produced by microbial fermentation in the colon, exhibits antiproliferative activities in various cancer cell lines. The initial objective of the study was to assess whether the effect of sodium butyrate (NaB) on cell growth differed by p53 status of the cells. Four human colorectal adenocarcinoma cell lines were used: HT29 (p53 point mutation), Caco2 (p53 truncation), LS513 (p53 wild type), and Lovo (p53 wild type). NaB significantly inhibited cell growth in all four cell lines. NaB arrested HT29 and LS513 cells in G0/G1 and Caco2 and Lovo in G2-phase. A second objective was to determine whether NaB similarly affected the cyclin-dependent kinase inhibitor, p21WAF1/CIP1. In all cell lines, p21 mRNA levels were immediately elevated after NaB exposure, and p21 protein levels were increased within 6 h. NaB increased p21 promoter activity in both Caco2 and Lovo, suggesting p53 independence. NaB did not influence p21 mRNA stability. Although three DNase I hypersensitivity sites were identified in the region of the p21 gene, induction of p21 mRNA by NaB was not accompanied by relaxation of the chromatin in the region of the p21 gene.     

Dietary Fatty Acids from Leaves of Clerodendrum Volubile Induce Cell Cycle Arrest,Downregulate Matrix Metalloproteinase-9 Expression,and Modulate Redox Status in Human Breast Cancer

The antiproliferative effect of the fatty acid components of Clerodendrum volubile leaves as well as its antioxidant effect on MCF-7 and MDA-MB-231 human breast cancer cell lines were investigated. Fatty acids extracted from C. volubile leaf oil were subjected to gas chromatography mass spectrometry (GCMS) analysis. The cells were cultured and treated with the fatty acids for 48 h, after which the antiproliferation effect was ascertained via MTT assay and cell viability analysis using BD fluorescence activated cells sorting (FACS) Calibur. Cell cycle was analyzed by flow cytometry on FACS Calibur. Western blotting was used in determining expression of proteins in the cell lines. The treated cell lines were assessed for reduced glutathione level, catalase, superoxide dismutase, and lipid peroxidation. The fatty acids significantly inhibited cell proliferation, arrested G0/G1 phase, downregulated the expression of MMP-9, and attenuated oxidative stress in of MCF-7 cell lines but had little or no effect on MDA-MB-231 cell lines. These results indicate the therapeutic potential of the fatty acids components of the leaves of C. volubile on human breast cancer, which may be explored further in drug development.     

Effects of Soy Isoflavones on Apoptosis Induction and G2-M Arrest in Human Hepatoma Cells Involvement of Caspase-3 Activation,Bcl-2 and Bcl-XL Downregulation,and Cdc2 Kinase Activity

Genistein, biochanin-A, and daidzein, the predominant soy isoflavones, have been reported to lower the risk of cancer, but it is not known whether they protect against human hepatoma cancer. This study was designed to investigate their effects on cell growth, the cell cycle, and apoptosis induction in the human hepatoma cell lines, HepG2, Hep3B, Huh7, PLC, and HA22T. Genistein, biochanin-A, and daidzein inhibited growth of all five lines in a dose-dependent manner. DNA fragmentation studies and the TUNEL assay demonstrated that isoflavones caused tumor cell death by induction of apoptosis. Activation of caspase-3 and cleavage of the caspase-3 substrate, poly(ADP-ribose)polymerase, was seen in hepatoma cells after 24 hours' exposure to isoflavones. In addition, isoflavone cytotoxicity correlated with downregulation of Bcl-2 and Bcl-XL expression. Synergistic effects of the three isoflavones were observed on cell growth inhibition, apoptosis induction, and anti-apoptotic protein expression. Flow cytometry showed that genistein, but not biochanin-A or daidzein, induced progressive and sustained accumulation of hepatoma cancer cells in the G2/M phase as a result of inhibition of Cdc2 kinase activity. Coapplication of caffeine prevented this cell cycle arrest, but not apoptosis, showing that cell cycle arrest was not necessary for apoptosis. Furthermore, the isoflavones combination also had a significant tumor-suppressive effect in nude mice. These results suggest that isoflavones might be promising agents for the treatment of human hepatoma.     

Effects of soy isoflavones on apoptosis induction and G2-M arrest in human hepatoma cells involvement of caspase-3 activation,Bcl-2 and Bcl-XL downregulation,and Cdc2 kinase activity

Genistein, biochanin-A, and daidzein, the predominant soy isoflavones, have been reported to lower the risk of cancer, but it is not known whether they protect against human hepatoma cancer. This study was designed to investigate their effects on cell growth, the cell cycle, and apoptosis induction in the human hepatoma cell lines, HepG2, Hep3B, Huh7, PLC, and HA22T. Genistein, biochanin-A, and daidzein inhibited growth of all five lines in a dose-dependent manner. DNA fragmentation studies and the TUNEL assay demonstrated that isoflavones caused tumor cell death by induction of apoptosis. Activation of caspase-3 and cleavage of the caspase-3 substrate, poly(ADP-ribose)polymerase, was seen in hepatoma cells after 24 hours' exposure to isoflavones. In addition, isoflavone cytotoxicity correlated with downregulation of Bcl-2 and Bcl-XL expression. Synergistic effects of the three isoflavones were observed on cell growth inhibition, apoptosis induction, and anti-apoptotic protein expression. Flow cytometry showed that genistein, but not biochanin-A or daidzein, induced progressive and sustained accumulation of hepatoma cancer cells in the G2/M phase as a result of inhibition of Cdc2 kinase activity. Coapplication of caffeine prevented this cell cycle arrest, but not apoptosis, showing that cell cycle arrest was not necessary for apoptosis. Furthermore, the isoflavones combination also had a significant tumor-suppressive effect in nude mice. These results suggest that isoflavones might be promising agents for the treatment of human hepatoma.     

Identification of specific Hep G2 cell binding regions in Plasmodium falciparum sporozoite-threonine-asparagine-rich protein (STARP)

It has been demonstrated that Plasmodium falciparum sporozoite threonine-asparagine-rich protein (PfSTARP) is located on the sporozoite surface. This protein's non-overlapping consecutive peptides were synthesised and tested in Hep G2 cell binding assays. Twelve high activity binding peptides (HABPs) were identified in the resulting 31 peptides. Three were found in 5' non-repeat region (amino acids 41-80). Peptides 20546 (41VIKHNRFLSEYQSNFLGGGY(60)), 20547 (61SAALKLVNSKKSGTNVNVTKY(80)) and 20548 (81NSENTNTNNNIPESSSTYTN(100)) were located in the conserved amino terminal region, as well as peptide 20548 which shared the sequence with the M region (amino acids 85-134). Six HABPs were located in region 10 (Rp10) (STDNNNTKTI). HABPs 20569 (501TSDDELNKDSCDYSEEKENI(520)) and 20570 (521KSMINAYLDKLDLETVRKIH(40)) were found in 3' non-repeat region. All these HABPs showed saturable binding and presented dissociation constants between 18 and 219 nM. The number of binding sites per Hep G2 cell ranged from 45000 to 370000. High binding peptides' critical amino acids involved in Hep G2 cell binding were determined by competition binding assays. SDS-PAGE results showed that both peptides 20570 and 20547 had at least two different sets of 44 and 38 kDa HABP receptors on Hep G2 cells. Specific modification of peptide 20546 and 20570 critical binding residues rendered these peptides immunogenic in Aotus monkeys, inducing high antibody titres against sporozoites, as assessed by IFA.     

Glucose-mediated inactivation of AMP-activated protein kinase reduces the levels of L-type amino acid transporter 1 mRNA in C2C12 cells

Branched chain amino acids (BCAAs) have protective effects against muscle atrophy. Although plasma BCAA concentrations are higher in patients with diabetes than in healthy subjects, diabetes is related to sarcopenia. We hypothesized that high glucose concentration reduces the quantity of BCAA transporters, and consequently, the effects of BCAAs are diminished despite their high levels. We examined whether glucose reduces the expression of L-type amino acid transporter 1 (LAT1), which transports neutral amino acids, including BCAA, in C2C12 myocytes. Glucose reduced LAT1 mRNA level by 80% in the C2C12 cells, compared with that in the glucose-free control cells. Regarding LAT1-related transporters, glucose also reduced the level of sodium-dependent neutral amino acid transporter 2 mRNA, but not that of 4F2 heavy chain. Although fructose reduced LAT1 mRNA levels, 2-deoxyglucose exhibited low effectiveness in reducing LAT1 mRNA level; galactose and mannitol had no effect. These results suggest a relationship between ATP produced during glycolysis and LAT1 mRNA levels. In fact, the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin reduced LAT1 mRNA levels in the absence of glucose, whereas the AMPK activator 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside increased LAT1 mRNA levels even in the presence of glucose. Consistent with these findings, glucose reduced the levels of phospho-AMPKα (Thr172) compared with that in the glucose-free control. These findings indicate that glucose inactivates AMPK, leading to a reduction in LAT1 mRNA levels in the C2C12 cells. This glucose-induced reduction in LAT1 expression may explain the unresponsiveness to BCAA in the patients with diabetes.     

Effects of branched-chain amino acid supplementation on plasma concentrations of free amino acids, insulin, and energy substrates in young men

The present study was conducted to examine alterations in the concentrations of plasma free amino acids, glucose, insulin, free fatty acids (FFAs), and urea nitrogen induced by branched-chain amino acid (BCAA) supplementation in young men. Overnight-fasted subjects ingested drinks containing 1 or 5 g of a BCAA mixture (weight ratio of 1 : 2.3 : 1.2 for isoleucine : leucine : valine), and blood was intermittently collected for 3 h after ingestion. Ingestion of the BCAA mixture resulted in significant increases in the plasma concentrations of individual BCAAs, corresponding to the amounts of amino acids ingested. On the other hand, plasma concentrations of methionine and aromatic amino acids tended to decrease in the trial with 5 g BCAAs, suggesting that BCAA ingestion affects the metabolism of these amino acids. The ingestion of BCAAs temporarily increased plasma insulin levels and affected plasma concentrations of FFAs, but had almost no effect on glucose or urea nitrogen.     

姜黄素通过Notch途径抑制人宫颈癌细胞株HeLa细胞增殖

目的:探讨姜黄素对人宫颈癌细胞株HeLa细胞体外增殖抑制及凋亡诱导作用及相关机制。方法:MTT法检测不同浓度姜黄素对HeLa细胞的增殖抑制作用;RT-PCR法检测HeLa细胞Notch1、Notch2、Jagged1、Bcl-2、Bax基因表达情况。结果:姜黄素对HeLa细胞具有增殖抑制作用,且呈时间-剂量依赖性。随着姜黄素剂量的增加,Notch1、Notch2、Bcl-2mRNA的表达逐渐下降,Bax mRNA的表达逐渐升高,Bcl-2/Bax亦逐渐减小,Jagged1mRNA无明显变化。结论:姜黄素可能通过Notch信号途径改变凋亡蛋白的表达,抑制HeLa细胞增殖。     

Comparison of the generation of albumin-associated cytotoxic activity in supernatants from ethanol-containing cultures of human blood monocyte-derived macrophages and of two human hepatoma cell lines.

The rates of oxidation of ethanol to acetate by human blood monocyte-derived macrophages and the two human hepatoma cell lines PLC/PRF/5 and Hep G2 were studied. The average rates obtained were, respectively, 621, 447 and 596 nmol/h/mg cellular protein. Cultures of these three cell types, containing known quantities of cellular protein per flask, were incubated with 0 or 2 mg ethanol/ml for 72 h and the culture supernatants subjected to affinity chromatography on blue sepharose CL-6B. Pure albumin fractions obtained in this way were adjusted to the same optical density and tested for cytotoxicity against A9 cells. The data showed that the albumin fractions obtained from ethanol-containing macrophage cultures were considerably more cytotoxic than those obtained from ethanol-containing cultures of PLC/PRF/5 and Hep G2 cells. It appeared that, for a given quantity of ethanol metabolised, considerably more acetaldehyde was released extracellularly by macrophages than by the two hepatoma cell lines and that this acetaldehyde bound to albumin to form cytotoxic acetaldehyde-albumin complexes. The data raise the possibility that macrophages are an important source of extracellular acetaldehyde and circulating acetaldehyde-albumin complexes in vivo.     

IgG在人肝细胞肝癌中的表达及其临床意义

目的：研究肿瘤源性IgG在人肝细胞肝癌中的表达和分布情况及其与临床病理学指标的关系。方法：分别应用免疫组化二步法和原位杂交检测60例肝细胞肝癌组织及其周围正常肝组织中IgG蛋白和IgG mRNA的表达,分析IgG的表达水平与9项肝细胞肝癌的临床病理学指标之间的关系。结果：肝癌组织中IgG的表达明显高于其周围正常肝组织,且两者表达程度比较差异有统计学意义（P=0.001）;IgG在肿瘤组织中的异常高表达与肿瘤分化程度和pTNM分期呈负相关（r=-0.357,P=0.005;r=-0.296,P=0.021）,与血浆AFP浓度呈正相关（r=0.336,P=0.009）,与年龄、性别、肿瘤包膜的完整性、肿瘤大小、肿瘤个数和一年内是否复发或转移无相关性（P〉0.05）。结论：肝癌细胞所产生的IgG与肿瘤的分化、pTMN分期相关,可能作为辅助肝细胞肝癌病理学诊断和预后评估的一项重要指标。