甲壳胺诱导人肝癌HepG2细胞凋亡的实验研究

目的:通过体外实验探讨甲壳胺是否对人肝癌细胞HepG2具有生长抑制及诱导凋亡作用.方法:在HepG2细胞培养液中加入不同浓度的甲壳胺,培养48 h,于倒置相差显微镜下观察甲壳胺处理组及对照组细胞形态学变化;用流武细胞术(FCM)检测HepG2细胞的凋亡率,Western印迹检测甲壳胺处理组及对照组Bcl-2和p53蛋白...     

紫花牡荆素诱导人肝癌HepG2细胞凋亡的研究

目的：研究紫花牡荆素（Casticin）对肝癌HepG2细胞增殖抑制和凋亡诱导的作用,并探讨其作用机制。方法：用终浓度为0、0.5、1.0、2.0umol/L的Casticin作用于HepG2细胞,于12、24、48h后采用MTT法检测细胞增殖抑制率;Hoechst33342核染色,观察细胞形态学变化;24h后收集各组肝癌HepG2细胞,流式细胞术检测细胞周期及凋亡率;RT-PCR检测survivin mRNA表达。结果：MTT法检测显示,Casticin对肝癌HepG2细胞有增殖抑制作用,并存在浓度和时间依赖关系;Hoechst33342染色后,可见核染色质凝集,凋亡细胞呈致密浓染,与对照组相比,Casticin处理后凋亡细胞比例增加;Casticin作用24h后,细胞被阻滞于G2/M期,随药物质量浓度的增加,细胞凋亡率逐渐增加;RT-PCR结果显示,Casticin下调肝癌HepG2细胞survivin mRNA表达。结论：Casticin在体外对肝癌HepG2细胞有明显的增殖抑制和凋亡诱导作用,初步推断Casticin诱发肝癌细胞凋亡与其对survivin基因表达的抑制有关。     

四逆散对人肝癌HepG2细胞增殖、凋亡的影响及其机制

目的:探讨含四逆散药液血清对人肝癌HepG2细胞增殖、凋亡的影响及机制。方法:将人肝癌HepG2细胞分为5组,每组3个复孔。实验组细胞用五氟尿嘧啶(5-FU)或不同浓度的含四逆散药液血清处理48 h后,用倒置显微镜观察含四逆散药液血清处理后人肝癌HepG2细胞形态的变化; MTT法检测含四逆散药液血清对HepG2细胞生长的抑制作用;荧光染色和流式细胞术分别分析含四逆散药液血清对HepG2细胞凋亡的影响。Rho123染色法检测线粒体膜电位变化,Western blot检测细胞凋亡相关蛋白的表达。结果:与对照组比较,含四逆散药液血清处理人肝癌HepG2细胞后,细胞数量显著减少(P<0.01),形态发生改变,呈现典型的凋亡细胞形态; G1期细胞数明显增加,而G2期细胞数量显著减少(P<0.05); Bax、Caspase-3、-9和Cyt-c的表达显著升高,而Bcl-2的表达显著降低(P<0.05);随着含四逆散药液血清浓度增大,HepG2细胞线粒体膜电位显著下降(P<0.05)。结论:四逆散可以抑制HepG2细胞增殖,并通过线粒体途径诱导细胞凋亡。     

线粒体参与介导MOPDO抑制HepG2细胞增殖、诱导其凋亡的作用

研究新合成的一氧化氮供体--核苷衍生物6-甲基-4-(2-氟-3,5-二-O-苯甲酰基-1-β-D-呋喃阿拉伯糖)-[1,2,5]噁二唑[3,4-d]嘧啶-5(4H),7(6H)-二酮1-氧化物(MOPDO)抑制人肝癌细胞HepG2增殖、诱导凋亡的作用.以不同浓度的MOPDO作用于人肝癌细胞HepG2,MTT检测与相差显微镜观察相结合,分析MOPDO对HepG2细胞增殖的影响;膜联蛋白-V/PI双染检测细胞凋亡率;JC-1染色检测线粒体膜电位(△ψm)变化;硝酸酶法测定细胞培养液中NO的含量.结果发现,MOPDO以时间和剂量依赖性方式抑制HepG2细胞的增殖,细胞形态出现相应的变化;细胞凋亡率的增加呈时-效和量-效关系;JC-1染色显示,线粒体膜电位随着MOPDO剂量的增加降低;MOPDO释放的NO量呈剂量依赖性增加.表明线粒体可能参与介导MOPDO抑制HepG2细胞的增殖、诱导其凋亡的作用.     

二烯丙基三硫通过线粒体依赖性途径诱导人肝癌HepG2细胞凋亡

二烯丙基三硫(diallyl trisulfide,DATS)对多种肿瘤有抗癌作用,但机制尚不完全清楚．为探讨DATS对人肝癌细胞系HepG2细胞凋亡的影响,用丫啶橙/溴化乙锭(AO/EB)法观察细胞凋亡情况,在显微镜下计数凋亡细胞数．JC-1荧光染色观察线粒体膜电势变化．Western blot法检测细胞色素c蛋白分布,ELISA法检测caspase-3活性． 结果显示,DATS诱导HepG2细胞凋亡,用 50 μmol/L 与100 μmol/L DATS处理48 h,细胞凋亡率分别达到60.33%和93.67%,并引起HepG2细胞线粒体膜电势降低．Western blot显示,DATS能诱导胞浆细胞色素c增加,与此同时,线粒体细胞色素c减少,诱导HepG2细胞caspase-3活化．提示：DATS可通过降低线粒体膜电势,促进细胞色素c由线粒体膜释放到胞浆中,激活caspase-3途径诱导人肝癌细胞系HepG2细胞凋亡．     

灵芝肽诱导人肝癌HepG2细胞凋亡的研究

研究了灵芝肽(GLP)在体外对人肝癌HepG2细胞凋亡的影响,并初步探讨了其作用机制。结果显示,透射电镜下可见细胞染色质浓缩、聚集于核边缘成块状,形成典型的凋亡小体;GLP使HepG2细胞阻滞于G0/G1期,随着GLP浓度升高,其G0/G1期的细胞比例随之增加;同时细胞的早期、晚期和总的凋亡率亦均随之增加,存在剂量-效应关系;Western blotting检测结果显示,抑制凋亡基因bcl-2和survivin表达下调,而促凋亡基因p53表达上调,并且都存在剂量依赖性;细胞凋亡的关键蛋白酶caspase-3被激活,并且caspase-3酶活性与GLP浓度亦有剂量依赖性。提示GLP体外可诱导人肝癌HepG2细胞凋亡,其作用机制可能与bcl-2和survivin表达下调、p53表达上调及Caspase-3被激活有关。     

反义p73基因诱导人肝癌细胞HepG2的凋亡

目的：构建表达重组反义p73基因的重组逆转录病毒,观察其对人肝癌细胞HepG2的凋亡诱导活性,并进一步探讨其作用机制。方法：克隆p73基因的反义片段,重组法构成逆转录病毒载体pBabe-p73（pBP73）,以脂质体Lipofectamine2000将其转染293A细胞进行病毒包装;将逆转录病毒感染人肝癌细胞HepG2,用MTT法检测细胞生长抑制情况,Western blotting检测p73的表达;再分别用琼脂糖凝胶电泳和流式细胞仪检测肿瘤细胞的凋亡;最后检测p53,caspase-3和bcl-2蛋白的表达变化。结果  重组质粒pBP73经鉴定连接正确,其转染293A细胞后上清液中可得到病毒,滴度达5×107pfu;MTT检测见pBP73病毒组48和72h细胞抑制率高于对照组（45.1% vs. 5.3%,69.5% vs.17.3%,均p<0.05）。琼脂糖凝胶电泳出现典型梯形条带;流式细胞仪检测出现凋亡峰,于转染48h后达最高峰,其凋亡百分率高达20.47%;p73蛋白高表达组p53和caspase-3蛋白的表达亦有显著升高（p<0.05）,但bcl-2蛋白未见表达差异。结论：成功构建了p73逆转录病毒,反义p73基因在体外能够有效地诱导人肝癌细胞HepG2的凋亡,其可能机制是通过激活caspase-3而发生作用。     

天然提取物诱导人HepG2 细胞凋亡的研究进展

细胞凋亡是机体维持内环境稳定,更好的适应生存环境采取的一种死亡过程。细胞凋亡异常与肿瘤的发生、发展存在密切的关系。细胞凋亡的信号途径主要有死亡受体介导的外源性通路、线粒体介导内源性通路、内质网信号通路及MAPK信号通路。通过作用于凋亡信号通路上一些关键基因,诱导肿瘤细胞凋亡被认为是临床抗肿瘤治疗最有成效的治疗方法之一。研究已证实多种天然提取物作用于凋亡信号途径中一些重要因子可诱导细胞凋亡,并取得较好的抑制肿瘤增殖的效果。本文是关于细胞凋亡机制及各种天然提取物作用于凋亡通路上主要基因进行抗肿瘤治疗研究进展的综述。     

MAT2A基因小干扰RNA诱导人肝癌细胞凋亡的分子机制

为探讨甲硫氨酸腺苷转移酶2A(MAT2A)小干扰RNA对人肝癌细胞生长和细胞凋亡的影响及其机 制,采用脂质体转染法将MAT2A小干扰RNA质粒表达载体转染人肝癌细胞系Bel 7402细胞、HepG 2细胞和 HepG3B细胞.半定量RT PCR检测MAT2A mRNA表达,Western印迹检测MAT2A 蛋白质表达, M TT法观察MAT2A小干扰RNA对肝癌细胞生长的影响,流式细胞仪及DAPI染色检测siRNA对肝癌细 胞凋亡的影响.为探讨其作用机制, 进一步检测转染后肝癌细胞MAT的活性、MAT1A mRNA表 达及SAM、SAH含量.结果发现, MAT2A小干扰RNA特异性抑制人肝癌细胞MAT2A mRNA和蛋白质 的表达, 刺激MAT表达由MAT2A向MAT1A转变, 降低了肝癌细胞中MATⅡ活性（P<005） ,从而诱导肝癌细胞凋亡; MAT2A小干扰RNA诱导Bel－7402细胞、HepG 2细胞、 Hep 3B细胞凋亡 指数分别为19．3%±2．8%、22．8%±3．5%、21．8%±4．2%, 较对照组siRNA(凋亡指数为5 2%±19%)具有明显差异(P<005).DAPI染色显示, MAT2A小干扰RNA转染组可见多个细胞核 浓缩、碎裂成蓝色的小块状,染色质凝聚,形成典型的凋亡小体, 而对照siRNA转染组未发现典型的 凋亡小体.肝癌细胞的生长也受到抑制,MAT2A小干扰RNA转染Bel 7402细胞、HepG 2细胞 、HepG3B细胞72 h后,细胞生长抑制率达高峰,分别为39．62%、41．27%、38．84%.肝癌细胞 中SAM含量明显升高(P<001),而SAH含量改变不明显, SAM/SAH变化伴随SAM含量变化而改 变.提示靶向MAT2A基因的siRNA通过升高肝癌细胞中SAM含量,刺激MAT表达由MAT2A向MAT1A转变, 从而诱导肝癌细胞凋亡,抑制肝癌细胞生长.     

JKA97诱导HepG2凋亡及其分子机制研究

目的:研究药物JKA97对人肝癌细胞HepG2增殖的抑制作用及其分子机制.方法:采用MTT法观察药物JKA97对细胞增殖的影响;倒置显微镜观察细胞形态变化;流式细胞术检测细胞凋亡;Western blot方法检测线粒体融合蛋白mfn2表达水平变化.结果:药物JKA97抑制人肝癌细胞HepG2细胞增殖,5、10、20 μmol/L作用组24 h抑制率分别为34.26%、43.08%、54.02%;药物JKA97诱导人肝癌细胞HepG2凋亡,10、20 μmol/L JKA97作用HepG2细胞24h,细胞凋亡率分别为22.9%、72.9%;线粒体融合蛋白mfn2表达水平显著降低.结论:药物JKA97对人肝癌细胞HepG2生长具有明显的增殖抑制作用,诱导细胞凋亡;线粒体融合蛋白mfn2表达水平降低可能是其重要的分子机制之一.     

Molecular mechanisms of echinocystic acid-induced apoptosis in HepG2 cells

Echinocystic acid (EA), a natural triterpone enriched in various herbs, has been showed to have cytotoxic activity in some cancer cells, and is used for medicinal purpose in many Asian countries. In the present study, we found that EA could induce apoptosis in human HepG2 cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed at 45 microM for 24 h. Molecular data showed that EA induced the truncation of Bid protein and reduction of Bcl-2 protein. EA also caused the loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to cytosol. Moreover, EA could activate c-Jun NH(2)-terminal kinase (JNK) and p38 kinase, and JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB200235 could block serial molecular events of EA-induced apoptosis such as Bid truncation, Bcl-2 reduction, cytochrome c release, caspase activation, and DNA fragmentation in HepG2 cells. These findings indicate that JNK- and p38 kinase-mediated mitochondrial pathways might be involved in EA-induced apoptosis and enhance our understanding of the anticancer function of EA in herbal medicine.     

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

Molecular mechanisms of hepatic apoptosis

Apoptosis is a prominent feature of liver diseases. Causative factors such as alcohol, viruses, toxic bile acids, fatty acids, drugs, and immune response, can induce apoptotic cell death via membrane receptors and intracellular stress. Apoptotic signaling network, including membrane death receptor-mediated cascade, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, lysosomal permeabilization, and mitochondrial dysfunction, is intermixed each other, but one mechanism may dominate at a particular stage. Mechanisms of hepatic apoptosis are complicated by multiple signaling pathways. The progression of liver disease is affected by the balance between apoptotic and antiapoptotic capabilities. Therapeutic options of liver injury are impacted by the clear understanding toward mechanisms of hepatic apoptosis.     

Suillin from the mushroom Suillus placidus as potent apoptosis inducer in human hepatoma HepG2 cells

During the search of new anti-cancer agent from high fungi, the ethyl acetate extract of the mushroom Suillus placidus was found to exhibit a significant cytotoxic activity against human hepatoma HepG2 cells. With bioassay-guided fractionation, a cytotoxic component suillin was isolated from the extract. The anti-cancer effect of suillin was subsequently examined in 8 human cancer cell lines by using MTT assay. It is of interest to note that human liver cancer cells (HepG2 cells, Hep3B cells, and SK-Hep-1) were preferentially killed by suillin with an IC₅₀ of 2 μM in a 48 h treatment.Mechanistically. suillin was found for the first time to induce apoptosis in HepG2 cells as characterized by DNA fragmentation, phosphatidyl-serine (PS) externalization, activation of caspase-3, -8, -9, depolarization of mitochondrial membrane potential, as well as release of cytochrome c into the cytosol. Moreover, the apoptosis induced by suillin was suppressed by both caspase-8 and -9 inhibitors. Western blot analysis revealed significant increases in the protein levels of Fas death receptor, adaptor FADD protein, pro-apoptotic protein Bad and a decline of Bid. These results suggest that the induction of apoptosis by suillin is through both death receptor and mitochondrial pathways. Taken together, our results suggest that suillin might be an effective agent to treat liver cancer.     

Molecular mechanisms of cytolysis induced by human lymphokine-activated killer cells

It has been shown that lysis of tumor target cells caused by lymphokine-activated killers is possible both upon a direct contact and in the presence of isolated nongranular cytotoxic proteins. The contact of cytolytic lymphocytes with K-562 cells leads to Fas L activation on the lymphocyte membrane and secretion of a broad spectrum of soluble cytotoxic proteins immunologically related to Tag 7 described earlier. These proteins can form inactive complexes, which are reactivated upon heating and addition of ATP. The proteins induced discrete cytolytic processes in tumor cells, differing in the rate of cytolysis and the mechanism of the apoptotic signal transduction. Fast processes (revealed in 3 h) mediated by caspases, and slow ones (in 24 h) with the supposed involvement of mitochondria were detected. A scheme for the lymphokine-activated killer interaction with target tumor cells is proposed.     

抑制survivin表达增强人肝癌HepG2细胞对高线性能量转移射线的辐射敏感性

探讨了肿瘤细胞中survivin的表达对高线性能量转移(LET)射线辐射敏感性的影响.根据Gen Bank提供的survivin序列,合成特异性survivin-siRNA寡核苷酸,转染人肝癌HepG2细胞,抑制survivin的表达.发现siRNA转染后诱导了HepG2细胞G2/M期阻滞,增加了自发性和辐射诱导的细胞凋亡.在高线性能量转移(LET)碳离子辐照后,siRNA转染细胞的克隆存活率明显下降.这些结果表明survivin表达是HepG2细胞产生对高LET射线辐射抗性的关键因素.     

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells

The cytotoxicity and antioxidant activity on human hepa toma cell line HepG2 of three flavonoids homogenous com pounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accom panied by an increase of apoptotic cell death after 48 h of incubation. Quercetininduced cell apoptosis was shown to involve p53 and p21 upregulation, Cyclin D1, Cdk2, and Cdk7 downregulation. These results suggested that the in duction of G2/M arrest, apoptosis, and cell death by quer cetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.