Long-Spacing Collagen and Proteoglycans in Pathologic Tissues

Immunohistochemical and ultrastructural findings in two cases of fibrolamellar carcinoma of the liver and two cases of hepatocellular carcinoma of the common histologic type are described. Ultrastructural examination of both cases of fibrolamellar carcinoma revealed the presence of neurosecretory (NS) granules which were sparse in some cells and abundant in others. Many of the tumor cells had a distinct oncocytic appearance with abundant mitochondria. A portion of the glutaraldehyde-fixed neoplasm was processed for the uranaffin reaction (an ultrastructural cytochemical stain specific for the NS granules of neuroendocrine tissue). Abundant uranaffin-positive granules were found in the neoplastic cells of both cases of fibrolamellar carcinoma, whereas no uranaffin-positive granules were found in hepatocellular carcinoma of the common histologic type. There was no statistical difference in the mean diameter of the uranaffin-positive granules measured from both cases. Immunohisto-chemistry revealed the presence of neuron-specific enolase (NSE) and serotonin in one of the two cases of fibrolamellar carcinoma and no NSE staining in two cases of hepatocellular carcinoma of the common histologic type. These findings suggest that some liver tumors presenting histologically as fibrolamellar carcinoma may be neuroendocrine in nature.     

miR-148b/DUSP1调节巨噬细胞分泌细胞因子CD206促进肝癌的发生

目的 探讨miR-148b/DUSP1信号通路调节巨噬细胞分泌细胞因子CD206的表达对肝癌发生的影响.方法 利用全自动磁珠提取纯化系统提取外周血单核细胞并培养,用粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)和巨噬细胞集落刺激因子(macrophage colony stimulating factor,M-CSF)分别诱导生成M1型和M2型巨噬细胞,CD68、CD206进行表型鉴定,ELISA检测M1和M2型巨噬细胞分泌的细胞因子CD206的表达,CCK-8和Transwell实验检测巨噬细胞分泌细胞因子CD206对肝癌细胞(HepG2和Huh7细胞)增殖、侵袭、转移的影响,双荧光素酶报告基因系统验证miR-148b与DUSP1的靶向结合.结果 初步分离并鉴定M1和M2型巨噬细胞,M2型巨噬细胞分泌的细胞因子CD206促进肝癌细胞的生长和侵袭、转移,双荧光素酶报告基因证实DUSP1为miR-148b的靶基因,miR-148b/DUSP1信号通路促进肝癌的发生、发展,巨噬细胞标志物与肝癌患者的临床病理特征相关.结论 miR-148b/DUSP1信号通路影响巨噬细胞分泌的细胞因子促进肝癌发生、发展.     

Low and high linear energy transfer radiation sensitization of HCC cells by metformin

The purpose of this study was to investigate the efficacy of metformin as a radiosensitizer for use in combination therapy for human hepatocellular carcinoma (HCC). Three human HCC cell lines (Huh7, HepG2, Hep3B) and a normal human hepatocyte cell line were treated with metformin alone or with radiation followed by metformin. In vitro tests were evaluated by clonogenic survival assay, FACS analysis, western blotting, immunofluorescence and comet assay. Metformin significantly enhanced radiation efficacy under high and low Linear Energy Transfer (LET) radiation conditions in vitro. In combination with radiation, metformin abrogated G2/M arrest and increased the cell population in the sub-G1 phase and the ROS level, ultimately increasing HCC cellular apoptosis. Metformin inhibits the repair of DNA damage caused by radiation. The radiosensitizing effects of metformin are much higher in neutron (high LET)-irradiated cell lines than in γ (low LET)-irradiated cell lines. Metformin only had a moderate effect in normal hepatocytes. Metformin enhances the radiosensitivity of HCC, suggesting it may have clinical utility in combination cancer treatment with high-LET radiation.     

Cyto and genotoxicity of gold nanoparticles in human hepatocellular carcinoma and peripheral blood mononuclear cells

Engineered nanomaterials have been extensively applied as active materials for technological applications. Since the impact of these nanomaterials on health and environment remains undefined, research on their possible toxic effects has attracted considerable attention. It is known that in humans, for example, the primary site of gold nanoparticles (AuNps) accumulation is the liver. The latter has motivated research regarding the use of AuNps for cancer therapy, since specific organs can be target upon appropriate functionalization of specific nanoparticles. In this study, we investigate the geno and cytotoxicity of two types of AuNps against human hepatocellular carcinoma cells (HepG2) and peripheral blood mononuclear cells (PBMC) from healthy human volunteers. The cells were incubated in the presence of different concentrations of AuNps capped with either sodium citrate or polyamidoamine dendrimers (PAMAM). Our results suggest that both types of AuNps interact with HepG2 cells and PBMC and may exhibit in vitro geno and cytotoxicity even at very low concentrations. In addition, the PBMC were less sensitive to DNA damage toxicity effects than cancer HepG2 cells upon exposure to AuNps.     

Changes in the expression of melatonin receptors induced by melatonin treatment in hepatocarcinoma HepG2 cells

Abstract:  Hepatocellular carcinoma (HCC) is one of the most common cancers and its incidence is increasing worldwide. Melatonin, an indoleamine hormone, exerts anti-oxidant, immunomodulatory, anti-aging, and antitumor effects. Previous studies have shown that melatonin can act through specific receptors, including MT₁, MT₂, MT₃ receptors as well as a nuclear receptor belonging to the orphan nuclear receptor family. Recently, we have described their role in the oncostatic and pro-apoptotic effects of melatonin on HepG2 human HCC cells. However, the potential role of the different melatonin cellular receptors on its antiproliferative effects remains unknown. In the present study, we examined the effect of melatonin treatment on HepG2 human HCC cells, analyzing cell cycle arrest and melatonin receptor expression. Melatonin was administered for 2, 4, and 6 days at 1000 or 2500 μ m . Melatonin induced a dose- and time-dependent inhibition on cell proliferation. This treatment caused an alteration in the cell cycle, with an increase in the number of cells in G₂/M phase at both 1000 and 2500 μ m melatonin concentrations, and a significant increase on S phase cell percentage by the highest dose. Furthermore, increases in protein expression of MT₁, MT₃, and retinoic acid-related orphan receptor-α were found after melatonin treatments. These increases were coincident with a significant induction in the expression of p21 protein, which negatively regulates cell cycle progression. Our results confirm the antitumor effect of melatonin in HCC cells, suggesting that its oncostatic properties are related, at least in part, to changes on the expression of their different subtypes of receptors.     

海藻色素糖蛋白对小鼠肝癌细胞PCNA和Bcl-2蛋白表达的影响

目的研究麒麟菜海藻色素糖蛋白(SPG)对小鼠肝癌细胞PCNA和Bcl-2蛋白表达的影响。方法将50只皮下接种H22肝癌细胞株的小鼠随机分为5组,每组10只。高、中、低剂量SPG组分别每日经口灌胃给予SPG100、50、10mg/kg,肿瘤对照组给予等量生理盐水,连续10d。环磷酰胺组隔天腹腔注射环磷酰胺20mg/kg。取肝癌组织用MTT法测定各组肝癌细胞增殖活性,免疫组化法检测各组肝癌组织增殖细胞核抗原(PCNA)和凋亡相关蛋白Bcl-2表达水平。结果高剂量SPG组和肿瘤对照组的肝癌细胞增殖活性以吸光度表示分别为(0.711±0.028)和(1.135±0.032),差别有显著性(P<0.05)。PCNA和Bcl-2蛋白表达率在高剂量组分别为28.32%和16.78%,肿瘤对照组分别为72.78%和65.16%,差异均有显著性(P<0.05)。结论 SPG可降低小鼠肝癌细胞PCNA和Bcl-2表达水平,对肝癌细胞增殖有一定抑制作用。     

维甲酸对DENA诱发肝癌大鼠的T淋巴细胞亚群与NK细胞的影响

目的：本文探讨维甲酸抑制大鼠肝癌细胞增殖对T淋巴细胞亚群和NK细胞的影响。方法：设三个实验组,分别收集30个大鼠外周血样本,然后进行免疫荧光抗体标记。应用流式细胞仪（EPICS-XL）检测T淋巴细胞亚群和NK细胞的百分率。结果：对照组与健康组比较,CD4＋细胞数明显减少,CD8＋细胞数明显增多,CD4＋/CD8＋比值下降（P〈0.01）。维甲酸组与健康组比较,CD4＋、CD8＋和CD56＋细胞数均无显著性差异（P〉0.05）。结论：维甲酸抑制大鼠肝癌细胞增殖时对机体免疫功能的改善有一定影响。     

肝细胞癌患者血清瘦素表达及其临床意义

目的探讨肝细胞癌患者血清Lep的表达和临床意义。方法用ELISA检测30例健康对照、31例肝硬化和146例HCC患者血清Lep含量,分析Lep在HCC临床分期及预后的关系。结果HCC患者血清Lep水平与BMI高度相关,Lep水平（ng/ml）HCC组（19.16±8.65）与肝硬化组（34.62±13.87）和健康对照组（27.41±11.72）比较差异有显著性（P〈0.05）,且性别差异明显（P〈0.01）,女性约为男性的2倍。随HCC患者TNM分期增加和转移、复发,其血清Lep水平明显降低（P〈0.05）。结论血清Lep水平可作为判断HCC患者营养状况和预后的重要指标。     

丹参素钠对大鼠肝癌细胞及自杀基因旁观者效应的影响

目的:探讨丹参素钠对大鼠肝癌细胞及自杀基因旁观者效应影响。方法:丹参素钠与丙氧鸟苷(GCV)分别或共同作用于大鼠肝癌CBRH7919的tk-细胞,以及含5%tk 细胞的tk 和tk-混合细胞,MTT检测各组存活率,两两比较各组存活率的差异,用q值分析药物与自杀基因系统联合的相互作用是否为协同性。结果:丹参素钠12.5、25、50、100 mg/L作用于CBRH 7919细胞72h存活率分别为87.8±12.4%、46.0±6.4%、20.2±4.6%、8.9±1.2%;IC50为24mg/L。10mg/L和50mg/L组的丹参素钠联合5%tk /GCV组的细胞存活率比单纯丹参素钠或单纯自杀基因组明显低,两两比较差异有统计学意义(P<0.05),q值均>1.15。结论:丹参素钠有明显抑制癌细胞生长作用,并能协同性增强tk/GCV系统对癌细胞的杀伤作用,增强自杀基因旁观者效应。     

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.