Ⅰ-Sce I系统的建立及其在诱导肝癌细胞DNA双链断裂中的应用

目的 建立Ⅰ-Sce I系统并应用该系统制备DNA双链断裂(DSB)的HepG2细胞模型,为进一步研究DSB与HBV DNA整合奠定基础.方法 通过分子克隆技术构建携带Ⅰ-Sce I内切酶识别序列的pEGFP2真核表达载体,并将其转染入肝癌细胞株HepG2,经G418筛选出稳定转染株,随后瞬时转染表达归位内切酶Ⅰ-Sce I的质粒pCMV-3NSL-Ⅰ-Sce I.24 h后采用免疫荧光法和Western blot检测DNA双链损伤效应分子γ-H2AX的表达,了解DSB情况.结果 酶切鉴定和测序证实pEGFP2构建成功;Ⅰ-Sce I系统引入后HepG2细胞中的γ-H2AX表达明显上调,提示DSB细胞模型构建成功.结论 Ⅰ-Sce I系统能够成功地诱导HepG2细胞株基因组发生DSB,该系统有望为进一步探讨DSB是否为HBV整合的潜在靶位提供有效的研究工具.     

人肝癌细胞系HepG2在遗传毒物检测中的应用及其进展

外来化合物的体外遗传毒性实验常用于各种致癌物和致突变物的快速筛选.HepG2是一种分化好的人肝胚细胞瘤细胞系,保留了较完整的代谢酶及其活性.以HepG2细胞作为实验系统检测各种致癌及非致癌物,在多个观察终点均获得相应的阳性及阴性结果.     

siRNA干扰MDM2基因对人肝癌HepG2细胞增殖和凋亡的影响

目的将靶向MDM2的siRNA转染人肝癌HepG2细胞,观察转染后细胞内p21基因的表达变化及其对HepG2细胞增殖和凋亡的影响。方法将人工合成的针对MDM2基因的siRNA片段通过LipofectamineTM 2000转染人肝癌细胞HepG2。实验分为MDM2 siRNA转染组、阴性对照组、脂质体组、正常对照组。分别用RT-PCR和Western blot法检测各组细胞中MDM2、p21的mRNA和蛋白质的表达量,并用MTT法检测各组细胞的增殖活力,流式细胞仪检测细胞周期和凋亡的变化。结果与其他3组比较,siRNA转染组细胞中MDM2 mRNA及蛋白表达减少（P〈0.01）,而p21 mRNA及蛋白表达增高（P〈0.01）。HepG2转染MDM2 siRNA后,增殖能力减弱,凋亡显著。结论 MDM2基因可能通过影响p21控制肝癌细胞的生长。     

Tyrosine sulfation of proteins from the human hepatoma cell line HepG2.

[35S]Sulfate labeling of the human hepatoma cell line HepG2 showed it to contain many sulfated proteins of diverse molecular weight range. The isolation of tyrosine O-sulfate indicated the supernatant fraction to contain a 5- to 7-fold higher level than the cellular fraction at the end of a 24-hr incubation. The proteins in the supernatant fraction were immunoprecipitated and examined for sulfation. Of 15 proteins tested, 7 were found to be sulfated as indicated by [35S]sulfate incorporation into proteins separated by NaDodSO4/PAGE and detected by autoradiography. The 35S-labeled bands were excised from the dried gel and subjected to extensive Pronase hydrolysis and the hydrolysates were analyzed for tyrosine [35S]sulfate by a two-dimensional procedure combining high-voltage electrophoresis and thin-layer chromatography [Liu, M. C. & Lipmann, F. (1984) Proc. Natl. Acad. Sci. USA 81, 3695-3698]. Of the sulfated proteins, three--fibrinogen, alpha-fetoprotein, and fibronectin--were found to contain tyrosine O-sulfate. The simultaneous presence of carbohydrate-bound sulfate, however, could not be exactly determined, but the other four [35S]sulfate-containing proteins--alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 2-macroglobulin, and transferrin--did not reveal any tyrosine O-sulfate and might be sulfated on their carbohydrate moieties.     

奥沙利铂对人肝癌细胞株HepG2体外增殖的影响

奥沙利铂(Oxaliplatin，L-OHP)是第三代铂类化合物，其化学名为左旋反式二氨环己烷草酸铂，国际通用名为草酸铂。临床前研究表明该药对人和鼠多种肿瘤细胞均有抑制作用，且对各种顺铂耐药的肿瘤细胞株无交叉耐药。研究通过抗肿瘤药物奥沙利铂对人肝癌细胞株HepG2体外增殖的影响，为奥沙利铂应用于肝细胞癌的临床治疗提供理论依据。     

Effects of Terminalia arjuna bark extract on apoptosis of human hepatoma cell line HepG2

AIM:To investigate the effects of Terminalia arjuna(T.arjuna)extract on human hepatoma cell line(HepG2)and its possible role in induction of apoptosis.METHODS:Human hepatoma cells were treated withdifferent concentrations of ethanolic extract of T.arjunaand its cytotoxicity effect was measured by trypan blueexclusion method and lactate dehydrogenase leakageassay.Apoptosis was analyzed by light and fluorescencemicroscopic methods,and DNA fragmentation.Themechanism of apoptosis was studied with expressionof p53 and caspase-3 proteins.Glutathione(GSH)content was also measured in HepG2 cells after T.arjunatreatment.RESULTS:T.arjuna inhibited the proliferation of HepG2cells in a concentration-dependent manner.Apoptoticmorphology was observed in HepG2 cells treated with T.arjuna at the concentrations of 60 and 100 mg/L.DNAfragmentation,accumulation of p53 and cleavage ofprocaspase-3 protein were observed in HepG2 cells afterthe treatment with T.arjuna.The depletion of GSH wasobserved in HepG2 cells treated with T.arjuna.CONCLUSION:T.arjuna induced cytotoxicity in HepG2cells in vitro.Apoptosis of HepG2 cells may be due tothe DNA damage and expression of apoptotic proteins.Depletion of GSH may be involved in the induction ofapoptosis of HepG2 cells.     

顺铂和苦参素对人肝癌细胞株HepG2端粒酶活性的影响

观察顺铂、苦参素对人肝癌细胞株HepG2端粒酶活性的影响,进一步探讨两种药物的可能作用机制。采用人肝癌细胞株HepG2作为研究对象,MTT法检测不同浓度和作用时间的药物对细胞增殖的抑制作用;流式细胞仪分析细胞周期的分布及凋亡;PCR-ELISA法检测端粒酶活性的变化。两种药物均具有抗HepG2细胞增殖、阻滞细胞周期、诱导凋亡及抑制端粒酶活性的作用。对端粒酶活性的抑制可能是两种药物发挥抗肿瘤作用的机制之一。     

乙型肝炎病毒X基因下调miR-192对人肝癌细胞株HepG2凋亡的影响

目的 探讨乙型肝炎病毒X基因(HBx)通过调节人肝癌细胞株HepG2中miR-192的表达而抑制其凋亡的机制.方法 设立3个细胞组:稳定转染HBx基因的HepG2细胞(HepG2/HBx),稳定转染空载体pcDNA3.1的HepG2细胞(HepG2/pcDNA3.1)以及未作转染的HepG2细胞.用流式细胞术分析3个细胞组的凋亡率差异,用Taqman探针荧光定量PCR检测3组细胞中miR-192的表达水平.转染miR-192后,用流式细胞术检测HepG2细胞凋亡率的变化,同时用SYBR Green荧光定量PCR和Western blot检测细胞中p53、PUMA表达的变化.计量资料均数的比较用单因素方差分析.结果 HepG2/HBx细胞的凋亡率为2.37％±0.35％,较HepG2/pcDNA3.1、HepG2细胞(11.46％±0.69％、12.50％±0.66％)明显降低(F=171.722,P＜0.01).miR-192表达在HepG2/HBx细胞中为49.1％±5.9％,较HepG2/pcDNA3.1、HepG2细胞(98.0％±8.9％,100％)也明显下调(F=14.319,P＜ 0.05).转染miR-192后HepG2细胞的凋亡率(15.74％±1.17％)较转染相应阴性对照的HepG2细胞的凋亡率(10.74％±1.15％)显著升高(F=18.415,P＜0.05),同时,p53、PUMA基因在mRNA (953:1.68±0.12比0.90±0.09,F=43.115,P＜0.05 ; PUMA:1.66±0.10比0.98±0.06,F=22.541,P＜0.05)和蛋白质水平(p53:3.07比1,PUMA:2.13比1)的表达均显著上升.结论 miR-192促进HepG2细胞凋亡,HBx通过下调miR-192抑制HepG2细胞凋亡.     

人ZEB1基因真核表达载体的构建及其对肝癌细胞株HepG2增殖凋亡的影响

目的：探讨ZEB1基因与肝癌的关系,进一步揭示ZEBl基因在肝癌发生发展过程中的作用。方法：利用基因重组技术构建pCI-neo-ZEB1真核表达载体：脂质体介导转染技术转染肝癌细胞系HepG2。采用WesternBlot技术检测重组质粒的转染效率,CCK8比色法测定重组质粒转染对HepG2细胞体外增殖能力的影响,流式细胞术检测重组质粒转染后HepG2细胞凋亡率。结果：重组质粒经Nhel和Xbal双酶切、测序与ZEBl基因序列一致;利用脂质体介导将pCI-neo-ZEB1重组质粒转染HepG2细胞株,经WesternBlot技术检测ZEB1基因在蛋白水平稳定高表达;与对照组比较,人HepG2细胞株转染pCI-neo-ZEB1真核表达载体后细胞增殖能力增强,细胞凋亡率明显降低。结论：成功构建重组质粒Pci-neo-ZEB1并转入肝癌细胞株HepG2后,细胞增殖能力增强,凋亡减少。     

Effects of dehydroepiandrosterone on gluconeogenic enzymes and glucose uptake in human hepatoma cell line, HepG2

Dehydroepiandrosterone (DHEA), the most abundant human adrenal steroid, improves insulin sensitivity and obesity in human and model animals. In a previous study, we reported that orally administered DHEA suppresses the elevated activities of hepatic gluconeogenic enzymes like glucose-6-phosphatase (G6Pase) in C57BL/KsJ-db/db mice. However, the molecular mechanisms by which DHEA ameliorates insulin resistance are not clearly understood. In the present study, we cultured the human hepatoma cell line HepG2 with DHEA and measured the enzyme activity and protein expression of G6Pase to investigate the direct effect of DHEA on glucose metabolism in hepatocytes. DHEA significantly suppressed both the activity and protein expression of G6Pase. Moreover, DHEA decreased the gene expression of G6Pase and phosphoenolpyruvate carboxykinase, both of which were maximal at 1 microM DHEA, whereas the mRNA level of glucose-6-phosphate translocase was unchanged. Furthermore, DHEA enhanced 2-deoxyglucose uptake, although its effect was much smaller than that of insulin. These results suggest that DHEA may act at multiple steps in the regulation of glucose metabolism in the liver.     

Establishment of a human hepatoma multidrug resistant cell line in vitro

AIM:To establish a multidrug-resistant hepatoma cell line(SK-Hep-1),and to investigate its biological characteristics.METHODS:A highly invasive SK-Hep-1 cell line of human hepatocellular carcinoma,also known as malignant hepatoma was incubated with a high concentration of cisplatin(CDDP) to establish a CDDP-resistant cell subline(SK-Hep-1/CDDP).The 50% inhibitory dose(IC50) values and the resistance indexes [(IC50 SK-Hep-1/CDDP)/(IC50 SK-Hep-1)] for other chemotherapeutic agents and the growth curve of cell...     

Influence of leptin and insulin on lipid transfer proteins in human hepatoma cell line, HepG2.

AIM: Phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein (CETP) are key enzymes in lipoprotein metabolism facilitating the transfer and exchange of cholesteryl esters, triglycerides and phospholipids between lipoproteins. In the study presented here, we investigated the influence of two hormones-the adipocyte-derived hormone leptin as well as insulin on the hepatic secretion of both, PLTP and CETP. METHODS: PLTP activity and CETP concentration-measured by exogenous substrate assay and enzyme-linked immunosorbent assay-were determined in supernatant of human hepatoma cell line HepG2 after single or combined exposure to leptin and insulin at physiological and supraphysiological concentrations, respectively. Messenger-RNA of PLTP and CETP was quantified by Northern blot analysis. RESULTS: Leptin suppressed PLTP activity and CETP-concentration by up to 33% and 23%, respectively. Insulin also suppressed PLTP activity by up to 11% and CETP-concentration by up to 16%. In combination, the two hormones had additive suppressive effects for both, PLTP activity and CETP-concentration. Northern blot analysis showed no difference in m-RNA levels after exposure to leptin or insulin. CONCLUSIONS: Leptin and insulin, both known to increase with body fat mass, suppress production of PLTP and CETP in HepG2 cells. When extrapolated to the in vivo situation, this suppressive effect may constitute a mechanism counteracting the potentially harmful action of lipid transfer proteins, particularly reduction of HDL-cholesterol, in conditions frequently associated with increased plasma triglyceride levels such as obesity and insulin resistance.     

pEGFP-prohibitin真核表达重组质粒的构建及其在人肝癌细胞系HepG2中的表达

目的构建pEGFP-prohibitin真核表达质粒,并鉴定其在pEGFP-prohibitin转染的人肝癌细胞系HepG2中的表达。方法采用高保真PCR扩增获得prohibitin的全长编码序列,构建真核表达载体pEGFP-prohibitin,并通过基因测序和BLAST比对鉴定重组质粒;提取pEGFP-prohibitin和pEGFP-N1质粒,并通过TurboFect转染试剂转染HepG2细胞;采用实时PCR和Western印迹方法分别在mRNA水平和蛋白质水平检测prohibitin蛋白在转染后HepG2细胞中的表达。结果成功构建pEGFP-prohibitin真核表达重组质粒,并将其成功转染HepG2细胞;pEGFP-prohibitin转染组细胞prohibitin的表达量比空载转染组和未转染组细胞明显增加。结论成功构建真核表达载体pEGFP-prohibitin,并证明抗增殖蛋白prohibitin在pEGFP-prohibitin转染人肝癌细胞系HepG2中高表达。     

TSA对人肝癌HepG2细胞增殖、凋亡及FHIT表达的影响

目的观察曲古抑菌素A（TSA）的体外抗人肝癌HepG2细胞作用及机制。方法取人肝癌HepG2细胞培养至对数生长期后随机分为TSA组、对照组、空白组,TSA组加入TSA至终浓度分别为125、250、500、1 000、2 000nmol/L,对照组加入等量二甲基亚砜（DMSO）,空白组只加培养基、无细胞,每组设6个复孔,作用24~72 h后MTT比色法测定HepG2细胞增殖抑制率（IR）,倒置显微镜和电镜观察HepG2细胞形态变化,TUNEL法检测细胞凋亡率,实时荧光定量PCR检测脆性组氨酸三联体（FHIT）基因表达,Western blot检测FHIT蛋白表达。结果与对照组相比,TSA组IR随TSA浓度增加和作用时间延长而增加（P〈0.05）,透射电镜下TSA组HepG2细胞出现凋亡早期改变;与对照组比较,TSA组细胞凋亡率升高,FHIT mRNA及蛋白表达增强（P均〈0.01）。结论 TSA体外能有效抑制肝癌细胞株HepG2生长并促进其凋亡,机制可能为上调FHIT表达。     

甲胎蛋白表达缺失的人肝癌HepG2细胞株的构建

目的：建立甲胎蛋白（AFP）表达缺失的HepG2细胞株。方法：选择AFP的RNA干涉靶序列,体外合成两段互补的寡核苷酸,与线性化的pll3．7连接,扩增纯化得到所需质粒。鉴定后将质粒用慢病毒包装、纯化得到病毒粗提液,与HepG2细胞共培养,转染后用RT—PCR和Westernblot实验检测细胞AFP的表达情况。结果：RT-PCR和Westernblot实验显示,干扰后HepG2细胞AFP基因和蛋白的表达显著减少。干扰效率达84％。所得细胞株传代后仍仅能检测到微量AFP表达。结论：成功构建稳定的AFP表达接近缺失的HepG2模型细胞株。     

PARP-1抑制剂PJ34对人肝癌细胞株HepG2的抑制作用

目的:研究聚腺苷酸二磷酸核糖转移酶-1(PARP-1)抑制剂PJ34对人肝癌细胞株HepG2增殖的影响及其作用机制,以及PJ34是否进一步增强γ射线对肝癌细胞的增殖抑制作用.方法:细胞增殖实验观察不同浓度的PJ34,以及PJ34合并γ射线照射对HepG2细胞增殖的影响,流式细胞仪检测PJ34对HepG2细胞凋亡的影响.结果:PJ34对HepG2细胞的增殖有显著的抑制作用(t=15.175,P<0.01).随着PJ34浓度的增加,其抑制作用进一步增强.1Gy的γ射线照射对HepG2细胞的增殖有明显抑制作用,但γ射线照射联合PJ34与单用PJ34或γ射线照射对肝癌细胞的增殖抑制作用相比较无明显统计学差异(t=-1.413,P>0.05).PJ34能诱导HepG2细胞凋亡,72h时凋亡率明显高于对照组,二者有显著性差异(33.2% vs 11.4%,P<0.01).结论:PARP-1抑制剂PJ34通过诱导HepG2细胞凋亡抑制人肝癌细胞的增殖;PJ34并不显著增加γ射线对HepG2细胞增殖的抑制作用.     

两种聚腺苷二磷酸核糖聚合酶1抑制剂对人肝癌细胞株HepG2增殖和凋亡的影响

目的观察聚腺苷二磷酸核糖聚合酶(PARP)-1抑制剂AG-014699和AZD2281对人肝癌细胞株Hep G2细胞抑制作用和凋亡,初步探讨PARP-1抑制剂诱导Hep G2细胞凋亡的机制,为肝癌提供一种新的治疗靶点。方法 MTT实验观察不同浓度的AG-014699和AZD2281对Hep G2细胞增殖的影响,用流式细胞术检测Hep G2细胞凋亡率;Western Blot法检测casepase3和casepase8蛋白表达水平。组间比较采用t检验。结果 AG014699和AZD2281均有抑制Hep G2细胞增殖的作用,且具有时间和浓度依赖性,但Hep G2细胞对两种PARP-1抑制剂的敏感性不同,用MTT法检测48 h AG-014699和AZD2281的IC50分别约为20、400μmol/L。因AZD2281不敏感,未做流式细胞术和Western Blot法检测细胞凋亡。用10、30、50μmol/L的AG-014699能诱导Hep G2细胞凋亡,48 h时凋亡率最高达(31.00±2.13)%,明显高于对照组(0.900±0.013)%,二者差异有统计学意义(P0.01)。30、50μmol/L的AG-014699作用Hep G2细胞48 h后的caspase3和caspase8蛋白水平相对于正常对照组显著增加。结论 PARP-1抑制剂AG-014699和AZD2281均能抑制Hep G2细胞增殖,但敏感性不同,AG-014699可诱导Hep G2细胞凋亡,通过上调caspase3和caspase8蛋白水平来诱导细胞凋亡。