紫薯花青素对油酸诱导HepG2细胞血脂代谢的影响

研究紫薯花青素对油酸诱导HepG2细胞血脂代谢的影响。MTT法检测细胞活力,筛选出合适的紫薯花青素浓度及油酸浓度,建立油酸诱导肝癌细胞(HepG2)高脂模型。分别以噻唑兰染色吸光度(MTT值)、Oil Red O脂滴染色、甘油三酯(TG值)、胆固醇(TC值)、脂肪代谢及胆固醇代谢基因SREBP-1C、FAS、ACC、SCD-1、HMGCR LDLR mRNA表达水平为指标,考察紫薯花青素对油酸诱导高脂细胞血脂代谢的影响。结果表明紫薯花青素能够显著抑制油酸诱导的HepG2细胞脂肪堆积作用,150μg/mL紫薯花青素能够显著降低脂肪和胆固醇合成相关基因的表达,推测其具有较好的降血脂生物活性。     

蚕蛹油多不饱和脂肪酸和α-亚麻酸对HepG2细胞胆固醇代谢的影响

目的探讨蚕蛹油多不饱和脂肪酸(SPO PUFAs)和α-亚麻酸(ALA)对油酸(OA)诱导的脂变HepG2细胞胆固醇代谢的影响及其机制。方法采用油酸诱导法建立脂肪变性肝细胞模型,分别用SPO PUFAs和ALA进行处理,油红O染色观察细胞内脂滴累积情况,酶法检测细胞内总胆固醇(TC)含量,ELISA检测细胞培养液中胆汁酸(TBA)含量,定量PCR检测LXRα、PPARγ以及ABCG1 mRNA表达。结果 0.5mmol/L油酸诱导HepG2细胞24h后,细胞内可见大量红色脂滴,细胞内胆固醇含量升高,SPO PUFAs和ALA干预后,细胞内的脂滴明显下降,细胞内胆固醇含量显著降低,细胞培养液中的胆汁酸显著升高,且呈剂量依赖性,高剂量组效果最明显。SPO PUFAs和ALA还能上调LXRα、PPARγ以及ABCG1 mRNA的表达。结论 SPO PUFAs和ALA能抑制HepG2脂滴积累,降低脂变HepG2细胞胆固醇含量,促进胆汁酸的转化,其机制可能与其通过PPAR/LXR-ABCG1细胞信号途径,促进细胞内胆固醇向胆汁酸转化并排出有关。     

高脂培养条件下槲皮素对大鼠肝细胞胆固醇代谢的影响

目的探讨槲皮素对大鼠肝细胞胆固醇代谢的影响及相关机制。方法灌胃给予大鼠高脂乳剂1w,建立大鼠高脂血症模型,实验结束后,无菌条件下眼眶取血,离心取血清。采用含0、10μmol/L槲皮素的10%胎牛血清培养液干预培养BRL肝细胞24h,换液,用含0、10μmol/L槲皮素的10%高脂血清培养液干预培养BRL肝细胞48h,测定培养液中总胆固醇(total cholesterol,TC)、游离胆固醇(free cholesterol,FCH)、胆固醇酯(cholesterol ester,CE)、低密度脂蛋白胆固醇(low-density lipoprotein cholesterol,LDL-C),细胞内TC、FCH、CE的含量和调节胆固醇代谢的蛋白质,以及关键酶的表达与活性变化。结果与对照组相比较,10μmol/L槲皮素组培养液中TC、FCH和CE的浓度较低,且差异有统计学意义(P0.05),槲皮素降低了LDL-C浓度,降低细胞内TC和FCH的浓度,升高细胞内CE含量,同时调节胆固醇代谢相应的酶和蛋白质,如羟甲基戊二酸单酰辅酶A还原酶(HMG-Co A Reductase,HMGCR)活性及其m RNA和蛋白质的表达水平较低,低密度脂蛋白受体(low-density lipoprotein receptor,LDLR)、胆固醇调节结合蛋白2(Sterol regulatory element-binding protein-2,SREBP-2)、三磷酸腺苷结合盒转运蛋白A1(ATP-binding cassette transporter A1,ABCA1)、肝X受体α(liver X receptorα,LXRα)m RNA和蛋白质的表达水平较高,且差异都具有统计学意义(P0.05)。结论在高脂环境下,槲皮素可通过调节HMGCR、LDLR SREBP-2、ABCA1和LXRα的表达来调节胆固醇的代谢。     

全谷豆复合包经SREBP-2、LDLR和Visfatin调控胆固醇代谢机制的探讨

目的探讨全谷豆复合包改善脂代谢紊乱大鼠胆固醇代谢的可能机制。方法 44只雄性SD大鼠随机分为高脂模型组、米面组、全谷豆复合包组和阴性对照组,分别给予对应饲料连续喂养8周。实验前后测定大鼠血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)及低密度脂蛋白胆固醇(LDL-C)含量;实验结束后股动脉取血,分离脏器称重,计算脏体比和脂体比,检测血清中Visfatin浓度、肝脏组织中SREBP-2 mRNA和LDLR mRNA的表达以及脂肪组织中VisfatinmRNA的表达。结果与高脂模型组和米面组相比,全谷豆复合包组大鼠体重、血清TC、TG、LDL-C水平显著下降(P<0.05),血清HDL-C水平显著升高(P<0.05)。全谷豆复合包组大鼠血清Visfatin水平显著低于高脂模型组和米面组(P<0.05);与高脂模型组和米面组比较,全谷豆复合包组大鼠肝脏组织中SREBP-2 mRNA和LDLRmRNA的表达显著升高(P<0.05),脂肪组织中Visfatin mRNA的表达明显降低(P<0.05)。结论全谷豆复合包可以改善高脂膳食诱导的脂代谢紊乱大鼠的血脂和Visfatin水平,可能的机制是全谷豆复合包能增加SREBP-2和LDLR的mRNA表达,降低Visfatin mRNA的表达,经SREBP-2、LDLR和Visfatin途径调控胆固醇的代谢,起到维持TC和LDL-C的正常水平,改善脂代谢紊乱的作用。     

甲醛对肝细胞极低密度脂蛋白分泌外排相关基因的影响

目的观察甲醛对肝癌细胞株HepG2细胞内外甘油三酯(TG)含量及细胞内极低密度脂蛋白(VLDL)分泌外排相关基因表达水平的影响。方法以0、0.004、0.02、0.1 mmol/L甲醛分别处理人肝癌HepG2细胞24、48 h,采用甘油磷酸氧化酶法(GPO-POD)测定细胞内外TG含量,采用QRT-PCR法检测细胞内载脂蛋白B100(apo B100)、微粒体TG转运蛋白(MTTP)、载脂蛋白E(apo E)、TG水解酶(CES3)、二酰基甘油酰转移酶1(DGAT1)、包被蛋白Ⅱ(COPⅡ)、Sar1b、低密度脂蛋白受体(LDLR)基因的表达水平。结果与阴性对照组相比,0.004、0.1 mmol/L甲醛染毒48 h后HepG2细胞内TG含量减少,染毒24 h后上清中TG含量增加;染毒24 h后0.004、0.02、0.1 mmol/L甲醛组或染毒48 h后0.004 mmol/L甲醛组细胞内apo B100基因表达水平升高;染毒24 h后0.004 mmol/L甲醛组或染毒48 h后0.004、0.02、0.1 mmol/L甲醛组细胞内MTTP基因表达水平升高;染毒24 h后0.1 mmol/L甲醛组细胞内LDLR基因表达水平降低,染毒48 h后0.02、0.1 mmol/L甲醛组细胞内LDLR基因表达水平升高;上述差异均有统计学意义(P0.05)。结论甲醛可能通过增加肝细胞内apo B100和MTTP表达来促进VLDL的分泌外排。     

非诺贝特对脂肪组织和细胞清道夫受体BI表达的影响

目的观察非诺贝特干预对高脂血症兔脂肪组织和细胞清道夫受体BI(SR-BI)表达的影响.方法 10只新西兰大白兔给予高胆固醇饮食饲养8周后,随机分为2组⑴高胆固醇组继续饲以高胆固醇饲料4周;⑵非诺贝特组在饲以高胆固醇饲料的基础上给予非诺贝特(30 mg/kg/d),共4周.另选兔(n＝5)普通饮食12周作为对照组.取腹股沟皮下脂肪组织行脂肪细胞培养.半定量逆转录多聚酶链式反应(RT-PCR)测定脂肪组织和细胞SRBI mRNA的表达.结果非诺贝特治疗组和高胆固醇组血清总胆固醇、低密度脂蛋白胆固醇水平均明显高于对照组(P＜0.001),但2组间差异无统计学意义 (P＞0.05).高胆固醇组兔脂肪组织和细胞SRBI基因表达水平较正常组增加(P＜0.05),非诺贝特组SRBI基因表达水平高于高胆固醇组(P＜0.05).结论非诺贝特能上调高脂血症兔脂肪组织和细胞SRBI mRNA表达, 提示非诺贝特可能参与脂肪细胞胆固醇代谢的调节.     

饱和脂肪酸与单不饱和脂肪酸对HepG2细胞自噬和凋亡的影响

目的比较饱和脂肪酸棕榈酸(palmitic acid,PA)和单不饱和脂肪酸油酸(oleic acid,OA)对HepG2细胞凋亡和自噬的影响。方法 PA和OA处理HepG2细胞,CCK-8检测脂肪酸的细胞毒性,caspase-3活性测定检测脂肪酸对细胞凋亡的影响,油红O染色检测细胞内脂质沉积,酶法检测细胞内甘油三酯的含量,Western blot检测自噬相关蛋白LC3-Ⅱ和凋亡相关蛋白caspase-3的表达,实时荧光定量PCR检测相关基因表达。结果 PA和OA都均同时诱导HepG2细胞凋亡与自噬,但PA的影响明显强于OA;PA诱导HepG2细胞自噬相关基因LC3B和VPS38表达、促进脂肪酸β-氧化的PPARα基因mRNA水平升高,但对细胞内脂肪沉积没有明显作用;而OA则导致HepG2细胞内甘油三酯的水平明显增加,但对上述基因表达没有明显影响。结论饱和脂肪酸PA和不饱和脂肪酸OA均能引起HepG2细胞凋亡和自噬,但PA的作用强于OA。     

金银花水提取总黄酮对高血脂小鼠的降血脂机制研究

目的探讨金银花水提取总黄酮(water extraction from honeysuckle total flavonoids, WEHF)对高血脂模型小鼠的降血脂作用及机制。方法应用高脂饲料建立高血脂模型小鼠的基础上,将50只小鼠随机分为正常对照组(10只)和造模组(40只)。造模组每天饲喂高脂饲料,正常组每天饲喂正常饲料,连续灌喂WEHF 28d。检测各组小鼠的体质量、饮食量、饮水量、血脂常数、肝脏指数、肾脏指数;检测肝脏内过氧化氢酶(catalase, CAT)活性、超氧化物歧化酶(superoxide dismutase, SOD)活性以及丙二醛(malondialdehyde, MDA)含量;检测胰岛素受体底物1(insulin receptor substrate, IRS1)、低密度脂蛋白受体(low density lipid receptor, LDLR)、载脂蛋白1(apolipoprotein 1, Apo-1)、脂肪酸合成酶(fatty acid synthase, FAS)、固醇调节元件结合蛋白(sterol regulatory element binding protein-1c, SREBP-2)和6-磷酸果糖激酶-2(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2, PFK-2)等mRNA表达;检测LDLR和SREBP-2的蛋白表达。结果模型组小鼠的体质量显著低于正常组(P0.05);饮食量和饮水量显著高于正常组(P0.05),且总胆固醇(Total cholesterol, TC)值为4.09mmoL/L,模型组与正常组小鼠的TC值有显著差异(P0.05),说明造模成功。与正常组比较,模型组肝脏指数显著上升而肾脏指数显著下降(P0.05)。与模型组相比,中、高剂量WEHF能显著降低高血糖小鼠血浆甘油三酯(Trilaurin, TG)水平(P0.05);高剂量WEHF的能显著降低高血脂小鼠血浆TC水平(P0.05);各WEHF组均能显著降低MDA含量(P0.05)和显著提高SOD活性(P0.05);WEHF中剂量、高剂量组能显著升高CAT水平(P0.05);高剂量的WEHF能显著上调IRS1、LDLR、Apo-1、FAS、SREBP-2和PFK-2等mRNA表达水平(P0.05);中、高剂量的WEHF能显著上调LDLR和SREBP-2的蛋白表达水平(P0.05)。结论 WEHF能改善高脂诱导小鼠肝脏的氧化应激水平,从而维持血液中正常脂质水平。其机制可能与WEHF上调模型小鼠肝脏IRS1、LDLR、Apo-1、FAS和SREBP-2等mRNA表达水平,上调LDLR以及SREBP-2转录因子的蛋白表达水平有关。     

染料木黄酮对HepG2细胞胆固醇代谢和SREBP-2通路的调控作用

目的探讨染料木黄酮（Gen）对HepG2人肝癌细胞胆固醇代谢和SREBP-2通路的调节作用。方法体外培养HepG2细胞,将HepG2细胞在含有0、0．01、0．10、1．00、10．00、50．00、100．00μmol／LGen的培养液内分别培养24、48、72h后,用MTT法检测细胞增殖活性。用分别含0、0．01、1．00、10．00、50．00μmol／LGen的完全培养液培养HepG2细胞24h,收集细胞,分别进行细胞内总胆固醇（TC）含量检测、实时荧光定量PCR检测细胞低密度脂蛋白受体（ldlr）、3-羟基-3-甲基戊二酸单酰辅酶A还原酶（hmgcr）基因的mRNA表达和蛋白印迹法检测核转录因子SREBP-2的表达。结果0．01、0．10、1．00、10．00、50．00、100．00μmol／LGen分别作用于HepG2细胞24h,0．01、0．10、1．00μmol／LGen组细胞增殖率高于溶剂对照组（均P〈0．05）;作用48、72h,1．00、10．00、50．00、100．00μmol／LGen组细胞增殖率均低于溶剂对照组（均P〈0．05）。1.00、10．00、50．00μmoL／LGen与HepG2细胞共同孵育24h,HepG2细胞内TC水平分别为（1．81±0．15）、（2．29±0．17）、（2．88±0．08）mmol／L,均高于对照组[（1．44±0．17）mmol／L],且随着Gen浓度升高,呈增加趋势（R2=0．48,P〈0．01）;各Gen实验组的hmgcr和ldlr基因的mRNA表达水平随着Gen浓度升高而升高（R2分别为0．53、0．79,均P〈0．01）。1．00、10．00、50．00μmol／LGen组蛋白表达灰度值均高于对照组（均P〈0．01）。结论染料木黄酮能够调节细胞内胆固醇的代谢,其作用机制可能与调控SREBP-2通路有关。     

全小麦纤维对载脂蛋白E基因敲除小鼠肝脏脂代谢的影响

目的 探讨全小麦纤维通过改善肝脏脂代谢发挥抗动脉粥样硬化(AS)的作用及机制。方法 20只7周龄雄性载脂蛋白E基因敲除(ApoE~(-/-))小鼠,按体重随机分为AS模型组和全小麦纤维组,同时选相同遗传背景的C57BL/6小鼠作为空白对照组。预防性干预18周后,取主动脉和肝脏进行HE染色,肝匀浆行总胆固醇(TC)、甘油三酯(TG)和游离脂肪酸(FFAs)测定;蛋白质印迹法(Western blot)测定肝脏组织中胆固醇调节元件结合蛋白-1(SREBP-1)、脂肪酸合成酶(FAS)、乙酰辅酶A羧化酶(ACC)以及SREBP-2、低密度脂蛋白受体(LDLR)和B族1型清道夫受体(SR-B1)的蛋白表达水平。结果 实验结束时,与空白对照组相比,AS模型组小鼠主动脉内膜明显增生,管腔内大量泡沫细胞和无结构样坏死物,全小麦纤维组小鼠管腔内泡沫细胞减少,粥样斑块面积明显减少;AS模型组小鼠肝脏有明显的肝细胞脂肪变性,全小麦纤维组小鼠肝脏脂肪空泡的数目明显减少,肝细胞脂肪变性有所改善;与AS模型组相比,全小麦纤维组肝匀浆TC水平降低[(60.56±13.49)μmol/g vs.(51.10±5.94)μmol/g](P<0.05);肝脏组织中SREBP-1、FAS、ACC的蛋白表达量降低(P<0.05),SREBP-2和SR-B1的蛋白表达量增加(P<0.05)。结论 全小麦纤维可通过调控参与肝脏脂代谢相关蛋白的表达,改善肝脏脂代谢,发挥抗AS作用。     

壁虎粉提取物作用于乙型肝炎病毒侵袭的HepG2和HL-7702细胞的实验研究

目的探讨壁虎粉提取物对乙型肝炎病毒侵袭过的人肝癌细胞(HepG2)和人肝细胞(HL-7702)中乙型肝炎病毒的复制能力和细胞生长状况的影响。方法实验分壁虎粉干预组、干扰素干预组、乙肝对照组和正常对照组四组,除正常对照组,其他三组以终浓度为1×107IU/mL含HBV-DNA的患者血清侵袭2小时后,再以一定浓度壁虎粉提取物或干扰素分别作用于上述两种细胞,收集各时段的培养液上清和(或)细胞,检测培养液上清AST、ALT、r-GT和LDH含量;采用荧光定量PCR法检测HBV-DNA;AO/EB进行细胞凋亡染色。结果两种细胞不同组别(除正常对照组外)培养6天、9天较培养3天AST显著增高(P<0.01),以两个干预组增高更为明显;HepG2干预组各时间段LDH含量高于HL-7702干预组(P<0.01),以壁虎粉干预HepG2组增高最为明显,同时凋亡细胞增多也较明显;两干预组细胞HBV-DNA含量较乙肝对照组低约1个数量级。结论壁虎粉提取物和干扰素对HBV复制具有抑制作用,对HL-7702细胞作用更为明显;壁虎粉提取物可诱导肝癌HepG2细胞早期凋亡、坏死。     

Ethanol Enhances Cholesterol Synthesis and Secretion in Human Hepatomal Cells

Excessive consumption of alcohol leads to severe alterations of lipid metabolism, including hyperlipemia and hypercholesterolemia. Following these epidemiological observations, we investigated the effects of ethanol at the cellular level by employing a human hepatomal cell line (HepG2) and by evaluating the biosyntheses of lipid classes from different labeled precursors. Incubation of cells with 2% ethanol resulted in a decreased labeling of phospholipids and in an increase in cholesterol synthesis and secretion. Triglyceride synthesis was increased by ethanol but their secretion in the medium was reduced, suggesting that these alterations may be related to their accumulation in the liver. The alcohol-induced alterations of lipid metabolism are not due to its metabolite acetaldehyde and data suggest that alcohol enhances cholesterol synthesis by affecting the initial steps without increasing HMGCoA expression. The observed modifications of lipid metabolism in HepG2 may partially explain the enhanced incidence of cardiovascular disorders that has been associated with alcoholism.     

Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids

The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomyelin, which promotes intracellular accumulation of ceramide. We have demonstrated for the first time that treatment with miltefosine strongly impedes the esterification of cholesterol and that this effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to higher levels of cholesterol in the cells. Indeed, miltefosine early impairs cholesterol transport from the plasma membrane to the endoplasmic reticulum, causing a deregulation of cholesterol homeostasis. Similar to miltefosine, other clinically-relevant synthetic alkylphospholipids such as edelfosine, erucylphosphocholine and perifosine show growth inhibitory effects on HepG2 cells. All the tested alkylphospholipids also inhibit the arrival of plasma-membrane cholesterol to the endoplasmic reticulum, which induces a significant cholesterogenic response in these cells, involving an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. Thus, membrane-targeted alkylphospholipids exhibit a common mechanism of action through disruption of cholesterol homeostasis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine and sphingomyelin biosyntheses certainly alter the ratio of choline-bearing phospholipids to cholesterol, which is critical for the integrity and functionality of specific membrane microdomains such as lipid rafts. Alkylphospholipid-induced alterations in lipid homeostasis with probable disturbance of the native membrane structure could well affect signaling processes vital to cell survival and growth.     

Conjugated linoleic acid upregulates LDL receptor gene expression in HepG2 cells

Conjugated linoleic acid (CLA) exerts anticarcinogenic and antiatherosclerotic effects in animals. The present study was conducted to examine the effects of CLA on LDL receptor (LDLr) expression in HepG2 cells, and to evaluate whether the sterol response element binding protein 1 (SREBP-1) and acyl CoA:cholesterol acyltransferase (ACAT) were involved in the regulation of LDLr expression by CLA. When HepG2 cells were cultured with serum-free DMEM for 48 h, there was a three- to fivefold (P<0.05) increase in LDLr protein and mRNA levels. Incubation of HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH, 5 mg/L) for 24 h decreased LDLr protein and mRNA by 50-70% (P<0.05) and mature SREBP-1 by 20-40% (P<0.05). CLA, but not linoleic acid, antagonized the depressive effects of 25OH and increased both LDLr protein and mRNA abundance twofold (P<0.05). LDLr protein and mRNA abundance were not different when HepG2 cells were cultured with CLA (0.4 mmol/L) plus 25OH in the presence or absence of an ACAT inhibitor (58-035, 1 mg/L). Furthermore, CLA had no effect on SREBP-1 abundance. These results suggest that CLA upregulates LDLr expression via a mechanism that is independent of ACAT and SREBP-1.     

Defining the Cholesterol Lowering Mechanism of Bergamot (Citrus bergamia) Extract in HepG2 and Caco-2 Cells

Bergamot, a Mediterranean citrus fruit native to southern Italy, has been reported to have cholesterol-lowering properties; however, the mechanism of action is not well understood. Due to structural similarities with 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitors, it has been proposed that the phenolic compounds in bergamot may also inhibit HMGCR. Statins are widely used for their cholesterol-lowering properties; however, they are not universally well tolerated, suggesting there is a need to identify novel cholesterol-lowering strategies. In the present study, we investigated bergamot fruit extract (BFE) and its principal components (neoeriocitrin, naringin, neohesperidin, melitidin, and brutieridin) for their ability to regulate cholesterol levels in HepG2 and Caco-2 cells. BFE at increasing concentrations decreased the levels of total and free cholesterol in HepG2 cells. BFE and its constituents did not directly inhibit HMGCR activity. However, BFE and neohesperidin decreased HMGCR levels in HepG2 cells, suggesting that neohesperidin and BFE may downregulate HMGCR expression. An increase in AMP-kinase phosphorylation was observed in BFE and neohesperidin-treated cells. In Caco-2 cells, brutieridin exhibited a significant reduction in cholesterol uptake and decreased the level of Niemann-Pick C1 Like 1, an important cholesterol transporter. Taken together, our data suggest that the cholesterol-lowering activity of bergamot is distinct from statins. We hypothesize that BFE and its principal constituents lower cholesterol by inhibiting cholesterol synthesis and absorption.     

柠檬酸镱诱导肝癌细胞HepG2凋亡作用及蛋白质表达变化研究

目的 研究稀土配合物柠檬酸镱([YbCit2]3-)诱导人肝癌HepG2细胞株凋亡的作用及其机制.方法 用DMEM培养基培养人肝癌细胞株HepG2细胞,以DMEM无血清培养细胞为对照组,以0.01～5.00 mmol/L[YbCit2]3-无血清培养基培养组为处理组,应用四噻唑蓝(MTT)法检测[YbCit2]3-对HepG2细胞生长的影响;以2.00 mmoL/L[YbCit2]3-无血清培养基培养组为处理组,Hoechst 33258染色法考察[YbCit2]3-对细胞的作用,采用差异蛋白质组学及H2O2、线粒体膜电位检测的方法研究[YbCit2]3-对HepG2细胞作用的可能机制.结果 2.00～5.00 mmol/L浓度范围内处理72 h,[YbCit2]3-能抑制HepG2细胞的生长,半数抑制浓度(IC50)值为(2.46 ±0.23)mmol/L.2.00 mmol/L[YbCit2]3-处理HepG2细胞48、72 h,Hoechst 33258染色检测表明其作用是诱导HepG2细胞凋亡.HepG2细胞经2.00 mmol/L[YbCit2]3-处理72 h后,通过双向电泳分离和质谱鉴定,鉴定出14个差异表达蛋白质点,包括丝切蛋白1、过氧化物氧化还原酶6、S100钙结合蛋白A6、蛋白酶26s非ATP酶亚基13亚型3等在细胞中分别参与抗凋亡、氧化还原、细胞增殖、蛋白降解等过程的蛋白质.2.00 mmol/L[YbCit2]3-分别作用HepG2细胞24、48 h后,细胞线粒体膜电位检测红绿荧光比值对照组为2.45 ±0.28,24 h组为1.56 ±0.23,48 h组为1.16 ±0.18,与对照组比,差异具有统计学意义(F=23.97,P=0.001);胞内H2O2检测荧光强度对照组为20.00 ±2.08,24 h组为40.00 ±5.50,48 h组为48.00±2.03,与对照组相比,差异具有统计学意义(F=48.40,P=0.000),表明[YbCit2]3-作用后线粒体膜电位下降,H2O2产生增加.结论 [YbCit2]3-可通过调节胞内氧化应激水平和线粒体凋亡途径诱导HepG2癌细胞凋亡.     

Long-chain polyunsaturated fatty acids upregulate LDL receptor protein expression in fibroblasts and HepG2 cells

The objective of this study was to investigate the effect of individual PUFAs on LDL receptor (LDLr) expression in human fibroblasts and HepG2 cells, and to evaluate whether acyl CoA:cholesterol acyltransferase (ACAT) and sterol regulatory element-binding protein 1 (SREBP-1) were involved in the regulation of LDLr expression by fatty acids. When fibroblasts and HepG2 cells were cultured with serum-free defined medium for 48 h, there was a 3- to 5-fold (P < 0.05) increase in LDLr protein and mRNA levels. Incubation of fibroblasts and HepG2 cells in serum-free medium supplemented with 25-hydroxycholesterol (25OH-cholesterol, 5 mg/L) for 24 h decreased LDLr protein and mRNA levels by 50-90% (P < 0.05). Arachidonic acid [AA, 20:4(n-6)], EPA [20:5(n-3)], and DHA [22:6(n-3)] antagonized the depression of LDLr gene expression by 25OH-cholesterol and increased LDLr protein abundance 1- to 3-fold (P < 0.05), but had no significant effects on LDLr mRNA levels. Oleic (18:1), linoleic (18:2), and alpha-linolenic acids [18:3(n-3)] did not significantly affect LDLr expression. ACAT inhibitor (58-035, 1 mg/L) attenuated the regulatory effect of AA on LDLr protein abundance by approximately 40% (P < 0.05), but did not modify the regulatory effects of other unsaturated fatty acids in HepG2 cells. The present results suggest that AA, EPA, and DHA increase LDLr protein levels, and that ACAT plays a role in modulating the effects of AA on LDLr protein levels. Furthermore, the effects of the fatty acids appeared to be independent of any change in SREBP-1 protein.     

Unsaturated fatty acids and phytosterols regulate cholesterol transporter genes in Caco-2 and HepG2 cell lines

Dietary consumption of phytosterols and certain fatty acids has been shown to reduce cholesterol absorption and plasma cholesterol concentrations. However, it has not been fully elucidated whether phytosterols or fatty acids can alter the expression of cholesterol transporters by functioning as signaling molecules. This study tested the hypothesis that various fatty acids and phytosterols commonly found in the food supply can modulate the expression of transporters including Niemann-Pick C1-like 1, low-density lipoprotein receptor, and scavenger receptor class B type I and 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the intestine and liver. Caco-2 cells were used as models of enterocytes, and HepG2 cells were used as a model of hepatocytes. The cells were treated for 18 hours with 100 μmol/L of a fatty acid, or for 24 hours with 10 μmol/L of 25α-hydroxycholesterol, or 100 μmol/L of cholesterol, sitosterol, and stigmasterol to measure expression of genes involved in cholesterol transport using quantitative real-time polymerase chain reaction. Polyunsaturated fatty acids in Caco-2 cells and sterols in HepG2 cells significantly reduced the messenger RNA expression levels of Niemann-Pick C1-like 1, scavenger receptor class B type I, low-density lipoprotein receptor, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Importantly, sitosterol and stigmasterol reduced the messenger RNA levels of genes to a similar extent as cholesterol. The data support the hypothesis that unsaturated fatty acid and phytosterols can act as signaling molecules and alter the expression of genes involved in cholesterol transport and metabolism.     

胸腺嘧啶核苷诱导肝癌HepG2细胞同步化

目的探讨胸腺嘧啶核苷（TdR）诱导肝癌HepG2细胞同步化的方法。方法于对数生长期的HepG2细胞中加入含终浓度为2．5mmoL／L的TdR培养28h后，PBS洗除TdR，加入新鲜血清培养基，此时记为0时刻，分别继续培养0、2、3、4、5、6、7、8、9、12、18、24、28h，收集细胞，同时实验设立对照组，采用流式细胞术检测细胞周期。结果分别于去除TdR后培养4、8、24h获得78．1％的S期细胞、67．2％的G2／M期细胞、86．3％的G1期细胞。结论终浓度为2．5mmoL／L的TdR处理肝癌HepG2细胞28h，再以新鲜培养基培养不同时间，可以获得同步化效果较好的S、G2／M和G1期细胞。