熊去氧胆酸选择性诱导人肝肿瘤细胞凋亡及抑制增殖的实验研究

目的探讨熊去氧胆酸(UDCA)诱导肝肿瘤细胞株凋亡并抑制其增殖的作用和机制。方法用噻唑蓝法、流式细胞术、TUNEL法、Wright-Giemsa染色法、电镜及免疫细胞化学等方法，观察UDCA对肝肿瘤细胞株HepG2、BEL7402和正常人肝细胞株L-02的生长活力、细胞凋亡、细胞周期及Bax／bcl-2基因表达的影响。结果UDCA对HepG2、BEL7402细胞株具有显著的抑制生长、诱导凋亡、阻滞细胞周期于S期、降低bcl-2和提升Bax表达的作用。结论UDCA对HepG2、BEL7402细胞株有显著的抑制增殖及诱导凋亡作用，该作用可能与UDCA阻滞细胞周期、降低bcl-2和提升Bax的表达有关。     

p38丝裂原活化蛋白激酶在顺铂诱导癌周肝硬化肝细胞株凋亡中的作用

目的 探讨p38丝裂原活化蛋白激酶(MAPK)通路在顺铂诱导正常肝细胞、癌周肝硬化肝细胞和肝癌细胞凋亡过程中的作用.方法 应用流式细胞仪、电镜、免疫细胞化学、激光扫描共聚焦显微镜和Western blot检测正常肝细胞株HL-7702、癌周肝硬化肝细胞株QSG-7701和肝癌细胞株QGY-7703加入顺铂和p38MAPK特异性抑制剂SB203580后的凋亡情况,以及p38MAPK、细胞分裂周期25同源体B(CDC25B)、p34cdc2和细胞周期素B1的表达.组间数据的比较采用单因素方差分析和SNK-q检验,P＜0.05为差异有统计学意义.结果 正常肝细胞、癌周肝硬化肝细胞和肝癌细胞株加顺铂后,凋亡率均明显增加,以癌周肝硬化肝细胞株最明显(从0.8%增高到41.5%);用顺铂+SB203580处理后,癌周肝硬化肝细胞株的凋亡率降低(28.1%),细胞周期阻滞于S期.电镜、免疫细胞化学、激光扫描共聚焦显微镜均观察到细胞凋亡形态.癌周肝硬化肝细胞分别用顺铂和顺铂+SB203580后,p38MAPK相对表达量分别为0.51±0.05和0.53±0.04,CDC25B相对表达量分别为0.61±0.04和0.59±0.03,p34cdc2相对表达量分别为1.08±0.16和1.21±0.15,与空白对照组(p38MAPK、CDC25B和p34cdc2的相对表达量分别为0.43±0.02、0.28±0.05和1.01±0.12)比较,差异有统计学意义(F=20.056,P＜0.05).结论 顺铂通过p38MAPK通路诱导癌周肝硬化肝细胞凋亡,而正常肝细胞和肝癌细胞的凋亡诱导途径可能不同.     

腺病毒介导MDA-7/IL-24选择性促进肝癌细胞的凋亡和增殖阻滞

目的 观察MDA-7／IL-24基因对不同p53状态人肝癌细胞HepG2、MHCC97L以及Hep3B和正常的肝细胞L02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和不同p53状态的肝癌细胞HepG2，MHCC97L、Hep3B。通过逆转录聚合酶链反应方法观察MDA7／IL24基因的表达，酶联免疫吸附法检测细胞培养上清液中MDA-7／IL-24蛋白的浓度，通过四甲基偶氮唑盐染色法及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和碘化丙啶双染后流式细胞仪检测细胞的凋亡，应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HepG2，MHCC97L和Hep3B以及正常细胞L02中高效表达。细胞培养上清液中有MDA-7／IL-24蛋白表达。MDA-7／IL-24能明显抑制各种肝癌细胞的生长，Hoechst染色提示MDA-7／IL-24促进肝癌细胞的凋亡，流式细胞仪提示MDA-7能选择性杀伤肝癌细胞而对正常的肝细胞无影响，细胞周期分析提示MDA-7／IL-24阻滞肝癌细胞在G2／M期，同时对正常的肝细胞没有促凋亡作用和增殖阻滞作用。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，选择性地杀伤肝癌细胞HepG2、MHCC97L和Hep3B，促进细胞增殖阻滞及诱导肿瘤细胞凋亡而与肿瘤细胞的P53基因的状态无关，同时对正常的肝细胞L02无任何毒性作用。     

没药甾酮对人肝癌细胞HepG2增殖和凋亡的影响

目的研究没药甾酮对人肝癌细胞HepG2增殖和凋亡的影响。方法以正常人肝细胞L-02作为对照,采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐法观察不同浓度没药甾酮（5～100μmol/L）对人肝癌细胞HepG2和L-02细胞增殖的影响并观察细胞形态的变化;应用流式细胞术检测细胞周期变化和凋亡发生。结果不同浓度没药甾酮均可显著抑制人肝癌细胞HepG2生长,并呈时间、剂量依赖性,最大抑制率可达81.9%±1.92%（100μmol/L）;没药甾酮可使G0/G1期细胞比例增多,G2/M期细胞比例下降,可将细胞阻滞于G0/G1期;没药甾酮诱导人肝癌细胞HepG2发生凋亡,50μmol/L和75μmol/L没药甾酮早期细胞凋亡率分别为24.91%±2.41%、53.03%±2.28%,与对照组相比,差异均具有统计学意义（P〈0.05）。结论没药甾酮可抑制人肝癌细胞HepG2增殖并诱导凋亡,其作用可能与干扰细胞周期有关。     

芒果甙对肝癌细胞增殖的抑制和凋亡的诱导

目的 观察芒果甙对人肝癌细胞株BEL-7404的毒性和诱导凋亡作用及对细胞周期的阴滞作用，探讨芒果甙作为肿瘤化学预防药物的可能性，方法 用MTT法观察芒果甙对人肝癌细胞株增殖的抑制作用。用光学显微镜观察芒果甙对细胞的毒性作用。以流式细胞仪检测芒果甙对细胞凋亡的诱导作用和对细胞周期的干预作用。结果 不同浓度的芒果甙在不同时间对肝癌细胞株均有毒性作用，并随浓度升高和作用时间的延长而毒性作用增强，凋亡也随之增加，芒果甙阻滞肝癌细胞周期于G2/M期，20μmol/L芒果甙作用24h后上述效果开始明显。结论 芒果甙对肝癌细胞株有明显的毒性作用。能诱导肝癌细胞凋亡和阻滞细胞周期于G2/M期具有潜在药用价值。值得进一步深入研究。     

去唾液酸糖蛋白-人端粒酶逆转录酶-胸苷激酶/丙氧鸟苷的靶向促HepG2细胞凋亡作用及旁观者效应

目的 观察去唾液酸糖蛋白(AF)-pGL3-人端粒酶逆转录酶(hTERT)-胸苷激酶(TK)对肝癌细胞株HepG2细胞生长及凋亡的影响.方法 培养细胞并构建pGL3-hTERT-TK质粒、AF脂质体复合物后,转染HepG2细胞和L02细胞,通过单光子液闪计数仪,脱氧核糖核苷酸末端转移酶介导的缺口末端标记法和流式细胞仪观察自杀基因对肝癌细胞生长和凋亡的影响,以及其对自杀基因的旁观者效应.结果 在肝癌细胞HepG2中,TK基因可以被hTERT启动子驱动高效的表达,而不影响正常肝细胞L02的生长,AF通过识别去唾液酸糖蛋白受体结合到HepG2细胞表面,其携带的TK基因更易进入肝癌细胞,同时增强自杀基因TK的高效表达,在旁观者效应机制的参与下,肝癌细胞总的凋亡率达85%±3%,而正常肝细胞则仅为16%±2%.结论 AF-pGL3-hTERT-TK可以靶向攻击肝癌细胞,对正常肝细胞几乎无影响,其基因传递系统具备靶向治疗肝癌的潜力.     

三种斑蝥素盐类物质对肝癌QGY-7703细胞生长的抑制作用

目的 考察斑蝥素酸镁、斑蝥素酸钠及斑蝥素酸钾,三种斑蝥素的盐类物质对肝癌QGY-7703细胞增殖的抑制作用.方法 采用WST-1比色法测定三种斑蝥素盐类物质对肝癌QGY-7703细胞的生长抑制率.结果 三种斑蝥素盐类物质对肝癌QGY-7703细胞的生长具有显著的抑制作用,抑制率随药物浓度升高其抑制作用增强,呈剂量效应关系.其半数抑制率IC50分别为9.48、33.80、35.04 μmol/L.结论 斑蝥素酸镁对肝癌QGY-7703细胞的抑制作用明显好于斑蝥素酸钠和斑蝥素酸钾,具有开发潜力.     

刺五加叶皂甙诱发肝癌细胞凋亡的研究

探讨刺五加（ASS）叶皂甙诱导体外培养肝癌细胞凋亡的作用。流式细胞仪检测ASS对HepG2 肝癌细胞的细胞周期动力学和凋亡诱导率的影响，并用维甲酸（RA）作为对照药和的。结果ASS和RA均作用于细胞周期的S期，抑制肝癌细胞的DNA合成。ASS还减少G2M期细胞数量，抑制细胞有丝分裂，诱发细胞凋亡率显著高于RA的作用，上述效应与其作用时间和剂量有关。ASS可促进体外培养肝癌细胞凋亡，有必要进一步探索ASS对肝癌的治疗价值。     

棘霉素诱导肝癌SMMC7721细胞凋亡的研究

目的研究棘霉素诱导肝癌SMMC7721细胞凋亡的作用,探讨其抗肿瘤的作用机制。方法通过细胞计数获得不同浓度棘霉素作用SMMC7721细胞的生长抑制率,观察棘酶素对肝癌细胞生长的影响;通过流式细胞仪分析棘霉素对细胞凋亡和细胞周期的影响。结果棘霉素浓度〉10 ng/ml时可抑制SMMC7721细胞的生长。结论棘霉素在体外可诱导肝癌细胞凋亡,具有很强的抗肿瘤作用。     

紫草萘醌类提取物通过抑制DNA合成诱导肝癌细胞凋亡

目的 研究紫草萘醌类化合物(LE)抑制肝癌细胞生长及诱导凋亡的机制.方法 MTT法测定LE对人肝癌细胞系SMMC-7721和人胚肾细胞系HEK-293的生长抑制作用;透射电镜检查、DNA电泳和原位末端标记检测凋亡;流式细胞仪分析细胞周期.结果 LE明显抑制人肝癌细胞SMMC-7721的生长.药物孵育24～72 h,电镜检查可见凋亡的典型过程;原位末端标记显示细胞核阳性染色.药物孵育16 h后流式细胞术检查:S期细胞明显减少.药物与正常传代细胞系HEK293孵育,对细胞周期没有影响.结论 LE具有专一性抗肿瘤作用,可诱导肝癌细胞凋亡.其作用机制为首先抑制DNA合成,进而引起细胞凋亡.     

人原发性肝癌细胞株受照射后凋亡与癌基因表达

目的 探讨人原发性肝癌细胞株受照射后凋亡与ｂｃｌ－２、ｐ５３基因表达产物关系。方法 选择人原发性肝癌细胞株ＱＧＹ,１８０ＫＶＸ线照射,流式细胞仪技术定量测定凋亡发生率,单克隆抗体免疫化法测定受照射后ｂｃｌ－２、ｐ５３表达产物,Ｓｏｎｙ Ｍｉａｓ－３００ Ｓｐａｔｏ图象分析仪分析结果。结果 ＱＧＹ－７７３０细胞株自发凋亡率为４．７９％,随照射后时间延长及照射剂量增加凋亡发生率增加。Ｂｃｌ－１、ｐ５３     

microRNA expression alteration after arsenic trioxide treatment in HepG-2 cells

Background and Aim: More and more microRNA (miRNA) are found to be involved in tumor genesis and progress. Arsenic trioxide has been an effective chemotherapeutic drug in cancer therapy for many years. In this study, we aimed to find the miRNA involved in the mechanisms of arsenic trioxide treatment in cancer therapy. Methods: We detected the expression profile of miRNA by miRNA microarray and quantitative real‐time polymerase chain reaction. Cell viability assay, flow cytometry analysis, prediction of miRNA targets, Western blot analysis and luciferase reporter assay were carried out to determine the role of one selected miRNA, namely mir‐29a, in affecting the biological behaviors of HepG‐2 cells. Results: Among the 677 human miRNA in the microarray, five miRNA were upregulated and four were downregulated in HepG‐2 cells treated with arsenic trioxide compared to their controls. If only changes above two folds were considered, four miRNA were identified, namely miR‐24, miR‐29a, miR‐30a and miR‐210, which were all upregulated. Among them, miR‐29a showed a positive therapeutic effect in liver cancer cells by inhibiting cell growth and inducing cell apoptosis, and PPM1D was confirmed to be the target gene of miR‐29a. Furthermore, a synergy effect was detected between miR‐29a and arsenic trioxide. Conclusions: Arsenic trioxide altered miRNA expression profile in HepG‐2 cells. Among the altered miRNA, miR‐29a seemed to take a role in the mechanism of arsenic trioxide in liver cancer therapy. The synergy effect between miR‐29a and arsenic trioxide may offer this drug a new chance in cancer therapy by decreasing its dose and toxic side‐effects.     

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.     

黑色素瘤相关抗原A1在肝癌细胞株中的表达与基因甲基化程度的相关性

目的 探讨人肝癌细胞株中黑色素瘤相关抗原A1(MAGE-A1)表达状况与肝癌细胞基因甲基化的关系。方法 提取10种人肝癌细胞株的总RNA,用逆转录聚合酶链反应对细胞株的MAGE-A1基因的表达进行检测;提取10种人肝癌细胞株的基因组DNA,用限制性酶切和Southern印迹杂交对每种细胞的基因组甲基化程度进行定量分析。另外对基因组DNA行HpaⅡ酶切后,用引物CDS21、EDP4和CDS20、EDP4进行聚合酶链反应扩增,然后用特异性探针杂交来检测肝癌细胞株MAGE-A1启动子的甲基化状态。用特异性序列聚合酶链反应方法检测每一种细胞株的人白细胞抗原-A位点型别。结果 QGY-7703、SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405肝癌细胞株的MAGE-A1基因表达阳性,且细胞分化程度均为中低分化;HepG2 2．2．15、HepG2、QGY-7701和Huh7肝癌细胞株的MAGE-A1基因表达阴性,且细胞分化程度均为高中分化。通过定量分析表明,MAGE-A1表达的肝癌细胞株与MAGE-A1不表达的肝癌细胞株比较,前者基因组甲基化程度较低(t=2．896,P=0．02)。肝癌细胞株MAGE-A1基因启动子区甲基化分析结果表明HepG2 2．2．15、HepG2、QGY-7701和Huh7为高甲基化,SMMC-7721、HLE、BEL-7402、BEL-7404、BEL-7405为低甲基化。结论 肝癌细胞株MAGE-A1 mRNA表达与其基因甲基化程度有关。     

HLA classⅠexpression in primary hepatocellular carcinoma

AIM：To investigate whetherCTL vaccine therapy is suitable for primary hepatocellular carcinoma(HCC)from the viewpoint of HLA classIantigens expression.METHODS:The immunocytochemistry,image analysis,flow cytometry,and labeled streptavidin bioti(LSAB)method of immunohistochemistry were applied respectively to study 4HCCcell lines(e.g.Alexander,HepG2,SMMC-7721,andQGY-7703)cultured in vitro and 6frozen tissue specimens of HCC.RESULTS:The positive control cell line Raji had very strong positive staining,Most mitotic and nonmitotic cells of the 4HCCcell lines had various intensity of HLAclassⅠantigens expression.The negative control cell K562 and the control slides of all the cell lines had no positive staining,In the 6HCCspecimens immunohistochemically studied,histological normal hepatocytes had no or very weak positive staining and the liver sinus had very strong positive staining.Most HCCcells in the sections from the 6HCCspecimens had strong positive HLAclassⅠantigens staining.The positive staining was located in the cytoplasm,the perinuclear area,and at the cell membrane of the liver cancer cells.Flow cytometry also revealed that Raji and those 4HCCcell lines had strong HLAclassⅠantigens expression.which was confirmed quantitatively by the image analysis.It showed that the objective grayscale values of Raji and those 4HCCcell lines were significantly different from that of K562(Raji114.04&#177;10.94,Alexander165.97&#177;5.35,HepG2167.02&#177;12.60,QGY-7703161.46&#177;7.13,SMMC-7721 165.93&#177;5.21,K562244.89&#177;4.60,P&lt;0.01).Significant differences were also found ebtween Raji and the 4HCCcell lines.CONCLUSION:HCC cells express HLA classI antigens strongly,Fromthis point of view.the active specific immunotherapy of CTL vaccine is suitable and practicable for HCC.     

三氧化二砷对人恶性黑色素瘤A375细胞增殖及新癌基因PPO表达的影响

目的 探讨三氧化二砷对人恶性黑素瘤A375细胞株细胞周期、增殖、凋亡及新癌基因PPO(proliferation and phosphorylation oncogene)表达的影响.方法 培养人恶性黑色素瘤A375细胞株,采用 MTT法检测三氧化二砷在不同浓度和不同时间下对A375细胞增殖的影响,流式细胞术分析三氧化二砷对细胞周期及凋亡的影响,倒置相差显微镜观察药物处理后对人A375细胞的细胞分化及形态的影响,免疫细胞化学(SP)法检测三氧化二砷对PPO蛋白表达的影响.结果 MTT法证实三氧化二砷在1～10 μmol/L浓度范围内对A375细胞有明显的增殖抑制作用,呈现明显的剂量和时间依赖效应关系.流式细胞术检测发现,随着药物浓度的增加,G0/G1的细胞逐渐减少(P＜0.01),S期的比例显著增加,出现S期阻滞.倒置显微镜观察发现细胞出现典型凋亡细胞形态学改变.免疫细胞化学法检测PPO蛋白主要表达于A375细胞胞浆内,染色呈棕黄色.三氧化二砷作用48 h后,随着药物浓度的增加,PPO的染色程度逐渐减弱,差异具有显著性(P＜0.01).结论 三氧化二砷能够显著抑制A375细胞株增殖,并诱导凋亡发生,且与三氧化二砷的浓度和作用时间成正相关,其作用机制可能是通过下调新癌基因PPO的表达发挥作用.