缺氧环境下三氧化二砷对肝癌细胞株BEL-7402生长及凋亡影响的研究

在人类实体肿瘤中，缺氧情况普遍存在。三氧化二砷（As2O3）在抗肿瘤治疗方面的主要机制为诱导肿瘤细胞凋亡。有研究证明，其对多种消化系肿瘤细胞的生长有抑制作用，但在模拟体内缺氧微环境条件下其对肝癌细胞的生长抑制作用及机制尚少有相关报道，本研究旨在探讨As2O3在缺氧环境下对肝癌细胞的生长抑制、诱导凋亡作用及其机制。     

三氧化二砷诱导多药耐药肝癌细胞BEL-7402/ADM凋亡的实验研究

目的研究As2O3诱导多药耐药肝癌细胞BEL-7402/ADM凋亡的效果,为As2O3在治疗已产生多药耐药性的原发性肝癌方面的应用提供理论依据。方法采用流式细胞仪检测、分析As2O3诱导BEL-7402/ADM、BEL-7402细胞凋亡情况。结果在体外As2O3能够诱导BEL-7402/ADM、BEL-7402凋亡,其强度与作用浓度和时间有关,两细胞系凋亡率差异无统计学意义。结论As2O3能够诱导表达mdr-1的多耐药细胞BEL-7402/ADM凋亡,将其应用于临床治疗已产生耐药性的原发性肝癌有可能产生较为理想的治疗效果。     

三氧化二砷对人恶性黑素瘤细胞株A375生长和凋亡影响的研究

目的研究三氧化二砷（As2O3）对人恶性黑素瘤细胞株A375的生长抑制与促凋亡作用。方法采用四甲基偶氮唑蓝（MTT）还原法检测As2O3对黑素瘤细胞的抑制作用；倒置显微镜、电镜观察细胞形态变化和凋亡特征；流式细胞仪分析DNA含量和检测细胞周期。结果1．0、2．0、4．0、8．0、16．0μmol／L As2O3均能抑制A375细胞的生长，且抑制率具有浓度和时间依赖性。8．0和16．0μmol／L组主要表现细胞毒性作用；4．0μmol／L组作用72h后，A375细胞出现较典型的凋亡形态特征，流式细胞仪检测到亚二倍体凋亡峰，同时出现S期阻滞。结论As2O3能有效的抑制人恶性黑素瘤细胞株A375的生长并促进其凋亡。     

三氧化二砷对正常肝细胞及肝癌细胞株的影响

目的观察不同浓度Ａｓ２Ｏ３作用不同时间对正常肝细胞及肝癌细胞株的影响。方法０．１２５～２μｍｏｌ／ＬＡｓ２Ｏ３与肝细胞及肝癌细胞株共孵育一定时间后,观察细胞的存活、形态学改变及细胞ＤＮＡ含量的分布。结果１μｍｏｌ／ＬＡｓ２Ｏ３处理的肝癌细胞第５天呈典型的凋亡特征性改变：在形态学上表现为细胞膜完整、染色质固缩、核碎裂、凋亡小体形成;流式细胞仪分析显示,在Ｇ１期细胞前出现亚二倍体峰,琼脂糖凝胶电泳呈凋亡特征性Ｌａｄｄｅｒ带;而正常肝细胞则未见明显改变。结论Ａｓ２Ｏ３可诱导肝癌细胞凋亡。     

缺氧诱发细胞凋亡的机制

细胞凋亡的发生主要有两条途径:细胞外的死亡受体途径及细胞内的线粒体途径.许多研究证实:缺氧组织内会发生不同程度的细胞凋亡失调,而凋亡失调是当今威胁人类健康的许多重大疾病的发病机制之一.老年人心血管疾病的患病率高,组织供血供氧不足较常见,因此了解组织缺氧致细胞凋亡的作用机制对一些老年性疾病的防治有重要意义.     

HIF-1α及其抑制剂对新生大鼠缺氧缺血性脑损伤时神经细胞凋亡的影响

目的 探讨缺氧诱导因子-1α(HIF-1α) 及其抑制剂2-甲氧基雌二醇(2ME2)对新生大鼠缺氧缺血性脑损伤时神经细胞凋亡的影响.方法 将120只新生7 d龄Wistar大鼠随机分为假手术组(Sham组,n=8)、缺氧缺血模型组(HIBD组,n=56)、2ME2干预组(2ME2组,n=56),后两组采用Rice法建立模型后,根据断头取脑的时间不同又分为3 h、6 h、12 h、24 h、48 h、72 h和7 d7个亚组.应用HE染色观察脑组织的病理变化,免疫组化技术检测HIF-1α、Bax、Bcl-2的表达,TUNEL法计数脑细胞凋亡.结果 HE染色显示2ME2干预后脑组织的损伤减轻;免疫组化显示:HIF-1α在HIBD组于模型制备后3 h升高,12 h达高峰,之后下降,2ME2组表达趋势和HIBD组相同,表达水平显著下降.与HIBD组比较,2ME2组各时间点的Bcl-2表达显著升高,Bax表达显著降低,TUNEL标记的阳性细胞数明显减少.结论 在新生大鼠缺氧缺血急性期使用2ME2抑制HIF-1α的表达,引起Bax/ Bcl-2表达量比值降低,凋亡细胞数目减少,改善脑损伤.     

CoCl2化学模拟肝癌体外缺氧模型的建立及对HIF-1α、VEGF基因的影响

目的观察不同浓度的氯化钴(CoCl_2)对人肝癌细胞生长及及其诱导化学性缺氧的最佳浓度和时间。方法通过不同浓度(0～800μmol/L)CoCl_2处理不同肝癌细胞系(HepG2、Hep3B、SMMC-7721和Bel-7402)24 h,利用CCK-8法检测四种人肝癌细胞株细胞存活率;选取100、200、300、400μmol/L CoCl_2作用不同肝癌细胞系0、12、24、48、72 h,利用CCK-8法检测四种人肝癌细胞株细胞存活率;确定最佳诱导浓度200μmol/L后,分别于0、6、8、12、24 h应用Real-time PCR检测VEGF mRNA的转录;确定好最佳诱导时间,采用Western-blot方法检测评估100、200、300、400μmol/L CoCl_2作用不同肝癌细胞系24 h后表达HIF-1α蛋白情况。结果 CoCl_2浓度在200μmol/L以内对人肝癌细胞株的生长无明显的抑制作用,当浓度大于200μmol/L、诱导时间大于24 h后明显抑制细胞生长,且抑制作用随着浓度的增加而增强(P0.05)。200μmol/L CoCl_2刺激四种肝癌细胞0、6、8、12、24 h时VEGF mRNA转录递增。200μmol/L CoCl_2刺激细胞24 h时,能诱导并稳定维持四种肝癌细胞HIF-1α蛋白表达。结论 CoCl_2诱导肝癌细胞化学性缺氧的最佳浓度为200μmol/L,此时最佳时间为24 h。     

三氧化二砷抑制人肝癌细胞株增殖和诱导凋亡作用

目的体外培养人肝癌细胞株HepG-2,BEL-7402,观察测定应用不同浓度三氧化二砷后多种指标的变化,从多个角度探讨三氧化二砷的抗肿瘤作用及其机制.方法应用倒置相差显微镜、电子显微镜、透射电镜、扫描电镜、流式细胞仪、细胞免疫组化法,分别对不同浓度加药组及对照组HepG-2,BEL-7402细胞的存活,形态学改变,细胞DNA含量的分布以及Bc1-2,Bax蛋白的表达进行了观察和测定.结果0.5,1,2μmol/L As2O3均能抑制人肝癌细胞株细胞的生长增殖.流式细胞仪分析显示,加药组在G1期细胞前均出现亚二倍体峰,且G0/G1期细胞减少,S期细胞增多,电镜下,对照组细胞核质比大、核大、核膜有明显切迹,0.5μmol/L As2O3组细胞核质比减小、核变圆、胞质内出现分化良好的细胞器,0.5,1,2μmol/L As2O3组均可见细胞膜完整、核固缩、凋亡小体形成.细胞免疫组化法测定Bcl-2,Bax蛋白的表达以及两者之间的比率均有变化.结论三氧化二砷不仅抑制人肝癌细胞增殖,而且诱导细胞凋亡.诱导肝癌细胞凋亡与Bd-2,Bax蛋白的表达改变有关.     

缺氧诱导因子-1α在脑缺血中的作用及其机制

缺氧诱导因子-1(hy poxia-inducible factor-1,HIF-1)是一种重要的转录调节因子,主要通过其活性亚基HIF-1α参与机体对缺氧环境的应答.脑缺血缺氧时,HIF-1α表达上调,可激活涉及糖酵解、血管生长、细胞生存和细胞凋亡的多个下游靶基因表达,对脑缺血后能量代谢障碍的改善以及微循环的建立具有重要意义.HIF-1α既可促进神经元存活,也可诱导神经元迟发性死亡.各种预处理和后处理方法可能通过激活HIF-1α调节神经细胞存亡.     

缺氧对人胰腺癌细胞整合素α5、β1表达的影响

目的探讨缺氧环境对人胰腺癌细胞株PANC1诱导因子-1α(hypoxia-inducible factor 1α,HIF-1α)、整合素α5(integrinα,INFα5)和整合素β_1(integrinβ,INTβ_1)表达及侵袭力的影响。方法以5%氧条件下培养PANC1,采用RT-PCR和Western blot检测HIF-1α、INTα_s、INTβ_1的mRNA和蛋白表达;采用Matrigel胶观测人胰腺癌细胞黏附侵袭能力的变化。并加入HIF-1α抑制剂YC-1缺氧培养PANC1,观察上述各指标的变化。结果HIF-1α蛋白表达从常规培养时0.497±0.011逐渐上升到缺氧培养24h的1.455±0.133(P<0.05),应用YC-1后蛋白表达下降到0.465±0.073(P<0.05),而HIF-1αmRNA表达无明显变化;INT_(α5)蛋白从常规培养时0.964±0.032逐渐下降到缺氧培养24h的0.465±0.073(P<0.05),应用YC-1后蛋白表达又上升到0.883±0.013(P<0.05).INT_(α5)mRNA的表达从0.212±0.023下降到0.018±0.002(P<0.05),应用YC-1后再上升到0.069±0.011(P<0.05);INTβ1的蛋白和mRNA表达于缺氧培养及YC-1处理后均无明显变化。细胞侵袭力从常规培养时69.9±30.5逐渐上升到缺氧培养24h的105.0±14.9(P<0.05),应用YC-1后又下降到79.7±20.9(P<0.05)。结论缺氧微环境下HIF-1α过表达可能通过下调INT_(α5)来调控胰腺癌细胞间和细胞与基质间的黏附能力,有利于进一步侵袭转移。     

Effect of arsenic trioxide on human hepatoma cell line BEL-7402 cultured in vitro

AIM:To study the effect of a varying concentrations of arsenic trioxide on human hepatoma cell line BEL-7402 cultured in vitro and its mechanism of action.METHODS:The BEL-7402 cells were treated with arsenic trioxide (at the concentrations of 0.5 1 2&mgr;mol/L, respectively) for 4 successive days. The cell growth and proliferation were observed by cell counting and cell-growth curve. Morphologic changes were studied with electronmicroscopy. Flow cytometry was used to assay cell-DNA distribution and the protein expression of Bcl-2 and Bax detected by immunocytochemical method.RESULTS:The cell growth was significantly inhibited by varying concentrations of arsenic trioxide as revealed by cell counting and cell-growth curve, which was dose- and time-dependent. Arsenic trioxide treatment at 0.5, 1 and 2&mgr;mol/L resulted in a sub-G1 cell peak, the apoptosis rate of the control group was 9.31% and that of 0.5&mgr;mol/L arsenic trioxide 15.53%, no significant difference was seen between the two.The apoptosis rates of 1,2&mgr;mol/L arsenic trioxide were 19.10% and 21.87% respectively, which were much higher (both P < 0.05). Decrease of G(0)/G(1) phase cells and increase of S phase cells were observed by flow cytometry, suggesting the inhibition effect of 0.5, 1, 2&mgr;mol/L arsenic trioxide on BEL-7402 cell lay in the G(0)/G(1) phase. Morphologic changes such as intact cell membrane, nucleic condensation, apoptotic body formation were seen under transmission electronmicrescopy, whereas the 0.5mol/L arsenic trioxide-treated BEL-7402 cells showed decrease of nucleocytoplasmic ratio, round nucleus, well-differentiated organelles in the cytoplasm. The processes and microvilli on the cell surface of the experimental groups under scanning electron microscopy were significantly decreased. High expressions of Bcl-2 and Bax were detected in 1 and 2&mgr;mol/L arsenic trioxide-treated cells, these were 46%, 87.33% and 83.08%, 95.83% respectively, among which that of Bax was more significant. Arsenic trioxide treatment at 0.5&mgr;mol/L resulted in a higher expression level of Bcl-2 and lower expression level of Bax,which were 8.81% and 3.83% respectively, as compared with that of the control group (15.33%) (P(1)<0.01, P(2)<0.01).CONCLUSION:Arsenic trioxide not only inhibited proliferation but also induced apoptosis of human hepatoma cell line BEL-7402. The induced-apoptosis effect of 1,2&mgr;mol/L arsenic trioxide was related to the expression level of Bcl-2 and Bax.     

The effect of arsenic trioxide on human hepatoma cell line BEL-7402 culturedin vitro

AIM To study the effect of a wide range of concentration of arsenic trioxide on human hepatoma cell lineBEL-7402 and its mechanism.METHODS The BEL-7402 cells were treated with arsenic trioxide (a final concentration of 0.5, 1 and2 μmol/L, respectively) in various durations or for 4 successive days. The cell growth and proliferation wereobserved by cell counting and cell-growth curve. Morphologic changes were studied under electronmicroscopy. Flow cytometry was used to assay cell-DNA distribution and the protein expression of Bcl-2 andBax was detected by immunocytochemical method.RESULTS The cell growth was significantly inhibited by the different concentrations of arsenic trioxide asrevealed by cell counting and cell-growth curve. Arsenic trioxide treatment at 0.5, 1 and 2 μmol/L, resultedin a sub-G1 cell peak. The decreased G0/G1 phase cell and the increased percentage of S phase cell were observed by flow cytometer, suggesting that the inhibiting effect of arsernic trioxide on BEL-7402 cell lay inG0/G1 phase cell. Apoptotis-related morphology, such as intact cell membrane, nucleic condensation,apoptotic body formation, can be seen under the electron microscopy. High protein expression level of Bcl-2and Bax was detected in 1 and 2 μmol/L arsenic trioxide-treated cells, but that of Bax was more significant.Arsenic trioxide treatment at 0.5 μmol/L resulted in higher expression level of Bcl-2 and lower expressionlevel of Bax compared with control (P1<0.01, P2<0.01).CONCLUSION Arsenic trioxide not only inhibited the proliferation but also induced apoptosis of humanhepatoma cell line BEL-7402. The induced-apoptosis effect of 1 and 2 μmol/L arsenic trioxide was relative tothe expression level of Bcl-2 and Bax.     

Peroxisome proliferator-activated receptor gamma ligands inhibit cell growth and induce apoptosis in human liver cancer BEL-7402 cells

AIM: To investigate the characteristics of PPAR gamma ligands induced apoptosis in liver cancer cells.METHODS: The effects of ligands for each of the PPAR gamma ligands on DNA synthesis and cell viability were examined in BEL-7402 liver cancer cells. Apoptosis was characterized by Hochest33258 staining, DNA fragmentation,TUNEL and EHSA, and cell cyde kinetics by FACS. Modulation of apoptosis related caspases expression by PPAR gamma ligands was examined by Western blot.RESULTS: PPARgamma ligands, 15-deoxy^-12,14-prostaglandin J2 (I5d-PGJ2) and troglitazone (TGZ), suppressed DNA synthesis of BEL-7402 cells. Both 15d-PG12 and TGZ induced BEL-7402 cell death in a dose dependent manner, which was associated with an increase in fragmented DNA and TUNEL-positive cells. At concentrations of 10 and 30μM,15d-PGJ2 or troglitazone increased the proportion of cells with Go/G1 phase DNA content and decreased those with S phase DNA content. There was no significant change in the proportion of cells with G2/M DNA content. The activities of Caspases-3, -6, -7 and -9 were increased by 15d-PGJ2 and TGZ treatment, while the activity of Caspase 8 had not significantly changed.CONCLUSION: The present results suggest the potential usefulness of PPAR gamma ligands for chemopreventJon and treatment of liver cancers.     

EGCG对氟尿嘧啶抑制肝癌细胞生长的增强作用

目的:研究表没食子儿茶素没食子酸酯(EGCG)是否具有增强5-氟尿嘧啶(5-FU)抑制肝癌BEL-7402细胞生长的作用,并探讨其中的机制.方法:应用MTT法观察肝癌细胞的生存率;Western blot进行蛋白分析:应用小RNA干扰(siRNA)法来沉默BEL-7402细胞中COX-2蛋白的表达;ELISA定量测定细...     

蟾毒灵对人肝癌BEL-7402细胞增殖的影响

目的：研究蟾毒灵对人肝癌BEL-7402细胞增殖及细胞周期的影响。方法：采用MTT法观察蟾毒灵对人肝癌细胞增殖的抑制作用；光镜及透射电镜观察细胞形态及超微结构；流式细胞术分析细胞周期分布情况。结果：蟾毒灵对肝癌细胞生长抑制作用呈浓度-时间依赖性，24、48和72小时的半数抑制浓度分别为6．67、0．348和0．228μmol／L，蟾毒灵可将细胞周期阻滞于G2／M期，透射电镜观察到典型的细胞凋亡特征。结论：诱导肝癌细胞周期G2／M期阻滞可能是蟾毒灵抑制肝癌细胞增殖的机制之一。     

三氧化二砷对体外培养的人子宫内膜腺癌细胞凋亡的影响

目的研究三氧化二砷(As2O3)对体外培养的人子宫内膜腺癌JEC细胞株细胞凋亡的影响。方法采用体外细胞培养的方法,以透射电子显微镜观察人子宫内膜腺癌JEC细胞株形态变化,流式细胞仪分析细胞DNA水平及细胞周期,并测定细胞凋亡情况。结果(1)4.0μmol/LAs2O3作用72h,在电子显微镜下可见细胞核固缩、核碎裂、染色质边集等改变;8.0、16.0μmol/LAs2O3作用48h后,部分细胞出现染色质边集及核碎裂;(2)As2O3<2.0μmol/L时,G0/G1期和S期细胞明显减少,G2/M期细胞增多;As2O3为2.0、4.0μmol/L时,在细胞周期G1期前可见典型的细胞凋亡峰;As2O3为8.0～16.0μmol/L时,则主要表现为细胞坏死。结论As2O3可诱导人子宫内膜腺癌JEC细胞株发生细胞凋亡。     

亚砷酸对肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响

目的探讨亚砷酸（AA）对人肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响。方法采用MTT比色法检测从作用后的BEL-7402细胞增殖抑制率,流式细胞术检测BEL-7402细胞周期及凋亡细胞,HE染色法观察凋亡细胞的形态,RT-PCR检测BEL．7402细胞的Bcl-2 mRNA,免疫组化法检测细胞的Bcl-2蛋白。结果1．0—8．0μmol／L的AA可使BEL-7402细胞增殖抑制率上升,能诱导BEL-7402细胞凋亡并阻滞细胞周期于S、G2／M期,呈剂量依赖性;8．0μmol／L的AA作用BEL-7402细胞48h后,细胞呈现明显的凋亡形态改变,其Bcl-2 mRNA及蛋白表达明显减弱。结论AA体外有抑制BEL-7402细胞增殖及诱导凋亡的作用,且呈时间、剂量依赖性,其作用机制可能与降低其Bcl-2表达有关。     

Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells

AIM:To study the effects of Pinus massoniana bark extract (PMBE) on cell proliferation and apoptosis of human hepatoma BEL-7402 cells and to elucidate its molecular mechanism. METHODS:BEL-7402 cells were incubated with various concentrations (20-200 ug/mL) of PMBE for different periods of time. After 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by morphological observation, agarose gel electrophoresis, and flow cytometry analysis. Possible molecular mechanisms were primarily explored through immunohistochemical staining. RESULTS:PMBE (20-200 ug/mL) significantly suppressed BEL-7402 cell proliferation in a time-and dose-dependent manner. After treatment of BEL-7402 cells with 160 ug/mL PMBE for 24, 48, or 72 h, a typical apoptotic "DNA ladder"was observed using agarose gel electrophoresis. Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. Sub-G_1 curves were displayed by flow cytometry analysis. PMBE decreased the expression levels of Bcl-2 protein in a time-dependent manner after treatment of cells with 160 ug/mL PMBE. CONCLUSION:PMBE suppresses proliferation of BEL-7402 cells in a time-and dose-dependent manner and induces cell apoptosis by possibly downregulating the expression of the bcl-2 gene.