朝鲜淫羊藿酸性多糖的理化特性及对HepG2细胞脂质堆积的改善作用＊

目的 对比研究朝鲜淫羊藿酸性多糖酯化还原前后的理化特性，并探讨其改善油酸诱导的肝癌HepG2细胞脂质堆积活性的差异。方法 采用高效凝胶渗透色谱法测定朝鲜淫羊藿酸性多糖（EFPA）的均一性和分子量，高效液相色谱法测定EFPA和酯化还原后朝鲜淫羊藿酸性多糖（EFPA-R）的单糖组成；采用油酸（OA）处理HepG2细胞诱导建立脂质蓄积模型，不同浓度EFPA与EFPA-R（10、30、100、300 μg·mL^-1）分别和OA共同作用于细胞24 h，采用CCK-8试剂盒测定细胞存活率，油红O染色观察细胞内脂滴蓄积情况，并采用试剂盒测定细胞内总胆固醇（TC）、甘油三酯（TG）含量。结果 EFPA为成分均一的多糖组分，分子量为125.8 kDa，由甘露糖、葡萄糖、半乳糖、葡萄糖醛酸和阿拉伯糖组成，摩尔比为1.7∶7.4∶1.4∶1.8∶1.0，葡萄糖占比最大，EFPA-R由甘露糖、葡萄糖、半乳糖和阿拉伯糖组成，摩尔比为0.8∶10.6∶2.1∶1.0；在10-300 μg·mL^-1范围内，EFPA和EFPA-R对HepG2细胞的抑制作用较弱，作为给药浓度；与空白组相比，模型组细胞中TC、TG含量显著升高（P < 0.01），细胞内红色脂滴显著增多，与模型组相比，EFPA可显著降低细胞中TC、TG含量（P < 0.01），明显减少细胞内红色脂滴（P < 0.05或P < 0.01），EFPA-R干预后细胞则无明显变化。结论 EFPA可明显改善HepG2细胞脂质堆积情况，且呈现剂量依赖性，而半乳糖醛酸（GalA）的存在可能是其抑制HepG2细胞脂质蓄积的关键因素。     

基于灰色关联度分析和聚类分析的3种滇产重楼抗肝癌作用谱效关系探索

目的:研究长柱重楼、滇重楼和南重楼的HPLC指纹图谱与其抗肝癌作用的谱效关系,为明确重楼抗肝癌作用的物质基础提供实验依据。方法:采用HPLC建立3种重楼提取物的指纹图谱,流动相乙腈(A)-水(B)梯度洗脱(0～10 min,20%A; 10～20 min,20%～25%A; 20～30 min,25%～30%A; 30～40 min,30%～35%A; 40～50 min,35%～40%A; 50～60 min,40%A; 60～75 min,40%～45%A; 75～80 min,45%～60%A),流速0. 9 m L·min~(-1),检测波长203 nm;利用噻唑蓝(MTT)比色法测定3种重楼提取物对肝癌HepG2细胞的增殖抑制作用,计算半数抑制浓度(IC_(50));运用聚类分析(HCA)和灰色关联度分析(GRA)研究3种重楼指纹图谱和抗肝癌作用的关系,找出对抗肝癌作用贡献较大的成分。结果:在3种重楼的HPLC指纹图谱中,确定其中11个色谱峰为共有峰。作用时间72 h时长柱重楼、滇重楼、南重楼的IC_(50)分别为148. 33,178. 87,208. 09 mg·L~(-1),其中长柱重楼的抗肝癌活性最强。灰色关联度结果显示,滇重楼共有峰中关联度较高的为1～10号峰,长柱重楼共有峰关联度较高的为1～7号峰,南重楼共有峰中关联度较高的为1～4,6～10,N1号峰,与IC_(50)关联度均0. 7。各重楼变量的聚类分析结果显示,可与IC_(50)聚为一类的色谱峰的关联度均 0. 7。结论:建立了3种重楼的HPLC指纹图谱,重复性良好。3种重楼中的1～4,6和7号色谱峰对抗肝癌药效贡献最大。     

腺病毒介导MDA-7/IL-24选择性杀伤肝癌 细胞HepG2的研究

目的 ：观察MDA-7/IL-24基因对肝癌的选择性杀伤作用，为肝癌的基因治疗提供理论基础。 方法 ：将携带人MDA-7/IL-24基因的腺病毒Ad.mda-7感染人正常肝细胞L02和肝癌细胞HepG2;用RT-PCR法观察MDA-7/IL-24基因的表达;ELISA方法检测细胞培养上清液中MDA-7/IL-24蛋白的浓度;4甲基偶氮唑蓝染色法（MTT）及Hoechst染色观察MDA-7/IL-24对肝癌细胞的生长抑制和杀伤作用;Annexin-V和PI双染后流式细胞仪检测2种细胞的凋亡;用流式细胞仪检测细胞周期。 结果 ：复制缺陷型腺病毒能介导外源基因MDA-7/IL-24在肝癌细胞株HepG2和正常细胞L02中的高效表达;细胞培养上清液中有MDA-7/IL-24蛋白的表达; MDA-7/IL-24能明显抑制肝癌细胞生长并可促进肝癌细胞的凋亡;MDA-7/IL-24阻滞肝癌细胞于G2/M期，能选择性杀伤肝癌细胞而对正常的肝细胞无阻滞作用和毒性作用。结论 ：复制缺陷型重组腺病毒载体Ad.mda-7能介导MDA-7/IL-24基因在人肝癌细胞中高效表达，促使细胞增殖阻滞及诱导肿瘤细胞凋亡，选择性地杀伤肝癌细胞HepG2，而对正常肝细胞L02无任何毒性作用。     

持续低剂量率照射后HepG2细胞的ATM磷酸化

目的 探讨持续低剂量率照射下HepG2细胞共济失调毛细血管扩张症突变基因(ATM)磷酸化的变化规律。方法 应用间接免疫荧光、Western blot技术检测持续低剂量率(8.28 cGy/h)照射下HepG2细胞ATM磷酸化蛋白的表达;采用集落形成法观察持续低剂量率照射对HepG2细胞增殖活性的影响。 结果持续低剂量率照射30 min后,ATM即已发生磷酸化,持续照射6 h时,ATM磷酸化蛋白表达量最多,以后逐渐减弱。使用Wortmannin抑制ATM磷酸化后,降低了持续低剂量率照射下肝癌细胞的存活分数。结论在持续低剂量率照射中后期ATM磷酸化减弱,提示持续低剂量率照射具有增加HepG2细胞辐射敏感性的潜能。     

Determination of phospholipidosis potential based on gene expression analysis in HepG2 cells.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.     

Influence of insulin on cholesterol removal from macrophages and cholesterol ester uptake by HepG2 cells

The fact that an increased blood insulin level is observed in patients with coronary artery disease (CAD) confirms the hypothesis that insulin promotes the development of atherosclerosis. The low high-density lipoprotein (HDL) concentration observed in such patients may contribute to alteration in reverse cholesterol transport and promote the accumulation of sterols in vascular tissue. We examined the effect of insulin (20−1000μUmL⁻¹) on cholesterol efflux into HDL₃ particles from human blood monocyte/macrophages and rat peritoneal macrophages preloaded with labelled cholesterol esters, and the influence of insulin on the accumulation of sterols by rat liver cells and HepG2 cell line in vitro models. Insulin at concentrations up to 250μUmL⁻¹ inhibited the efflux of cholesterol from rat macrophages and promoted high uptake of sterols by both types of hepatic cells. Pharmacological concentrations higher than 250μU mL⁻¹ exerted the opposite effect. In the case of human macrophages, an insulin concentration of 20μUmL⁻¹ increased cholesterol removal, whereas 100−200μU mL⁻¹ insulin inhibited cholesterol removal from cells, and very high concentrations (>350μUmL⁻¹) again increased cholesterol removal. We have shown that insulin excess counteracts the beneficial effects of HDL in removing cellular cholesterol and, therefore, may promote development of atherogenesis.     

紫丁香叶提取物对HepG2.2.15细胞中HBeAg及HBsAg表达的影响

目的 :观察紫丁香叶提取物对 Hep G2 .2 .1 5细胞分泌 HBe Ag和 HBs Ag的影响。方法 :采用酶联免疫吸附法 ( ELISA)检测培养上清中 HBe Ag和 HBs Ag表达的变化并以抑制率来表示。结果 :紫丁香叶提取物对 HBe Ag和 HBs Ag的分泌具有抑制作用 ,该作用呈现剂量依赖性和时间依赖性。结论 :紫丁香叶提取物对 HBe Ag和 HBs Ag分泌的抑制作用提示其在体外具有抗乙型肝炎病毒的作用     

过氧化氢对不同时相HepG2细胞氧化作用的研究

背景与目的:研究过氧化氢(H2O2)对不同时相HepG2细胞作用的选择性,以进一步探讨H2O2:诱导肿瘤细胞凋亡的机制.材料与方法:以胸腺嘧啶核苷(TdR)阻断法获得不同细胞周期时相的同步化细胞,分别加入800 μmol/L的H2O2作用3 h,并检测各组细胞的丙二醛(MDA)含量、黄嘌呤氧化酶(XOD)和5'核苷酸酶(5'NT)的活性及抗羟自由基(抗·OH)和抗超氧阴离子自由基(抗O2)的活性.结果:经H2O2处理的各组MDA含量和XOD、抗·OH、抗O2活性显著高于对照组,其差异均具有统计学意义(P均＜0.01),5'NT活性低于对照组(P＜0.01).与未同步化组比较,同步化于S期和G2/M期的细胞MDA含量和XOD、5'NT、抗·OH和抗O2活性显著升高,其差异均具有统计学意义(P均＜0.01),而同步化于G1期的细胞MDA含量、XOD、5'NT和抗O2-活性显著降低,差异均具有统计学意义(P均＜0.01).结论:H2O2作用同步化HepG2细胞后,代谢产生自由基的酶活性,脂质过氧化产物含量以及抗氧化水平均表现出细胞周期特异性,这可能是过氧化氢对不同时相HepG2细胞损伤作用不同的原因.     

顺铂持续压力下HepG2存活细胞中P53、C-myc、Bax、Bcl-2的变化及意义

目的动态观察HepG2细胞在顺铂压力下存活细胞中P53、C-myc、Bax、Bcl-2等的变化,进一步探讨HepG2顺铂耐药的机制.方法采用免疫组化法和RT-PCR法测定在顺铂压力下存活HepG2细胞内凋亡和抗凋亡因子P53、C-myc、Bax、Bcl-2、NF-κB的变化.结果在顺铂压力下存活HepG2细胞P53、C-myc等凋亡相关因子无明显变化.Bcl-2持续升高,Bax逐渐下降,Bcl-2/Bax的比率值呈进行性升高.NF-κB mRNA在7 d后出现明显阳性.结论在顺铂持续压力下HepG2细胞的存活机制与Bcl-2持续升高、Bax逐渐下降有关,NF-κB mRNA的阳性表达可能与细胞存活的后续事件有关.