Effects of adenoviral vector-mediated transduction of human p53,B7-1 and GM-CSF genes on liver cancer cells

The potential efficacy and clinical feasibility of gene therapy for liver cancer were tested through therecombinant adenovirus-mediated (Ad-multigenes ) co-transfer of human wild-type p53, B7-l co-stimulation(CD8o) and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes into human hepatocellular carcinoma cell lines. The treated cells underwent apoptosis with specific DNA fragmentation and became more sensitiveto cisplatin, a chemotherapeutic drug. Their growth was partly inhibited. Efficient proliferation and generation ofCTLs and cytokine production were induced in mixed lymphocytes through tumor cell reaction (MLTR) using peripheral blood T lymphocytes from donors as effector cells and Ad-multigenes or Ad-p53-transfected human hepatocellular carcinoma cells (HepG2 or BEL7402) as stimulator cells. Ad-multigenes-transfected rat carcinosarcomaWalker 256 cells were inoculated subcutaneously into normal rats. Fourteen days later, the activity of spleen cellsin rats inoculated with Ad-multigenes-transduced Walker 256 cells was higher than that in Ad-p53-transducedones. These findings suggest that adenovirus-mediated multigenes p53, B7-1 and GM-CSF can induce apoptosis ofliver cancer cells and initiate a potent antitumor immune response against them.

Modulating effects of survivin antisense oligonucleotide on changes of apoptosis and cell cycle of human hepatocellular carcinoma cell line SMMC-7721

To investigate the modulating effects of survivn antisense oligonucletode (ASODN) on the cell cycle and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and explore its mechanism.Methods Survivin ASODN was transfected into SMMC-7721 cells mediated by DOTAP liposomal reagent.Electron microscopy,flow cytometry and RT-PCR were used to detect the changes in cell ultrastructure,apoptosis,cell cycle and the expression of cyclinB1 mRNA,respectively.Results After transfection of survivin ASODN,the expression of cyclinB1 mRNA in the cells significantly increased and increase in G2-M arrest and apoptosis appeared.Meanwhile,the cell ultrastructure had apoptotic changes such as chromatin condensation and apoptotic body formation.Conclusion Survivin ASODN can induce the expression of cyclinB1 that may result in G2-M arrest.Consequently,apoptosis is triggered.Survivin ASODN transfection might be an improtant new treatment for HCC.14 refs,2 figs,1 tab.

Effects of cytotoxic T lymphocytes on hepatoma cell line SMMC-7721 induced by different subsets of dendritic cells in vitro

BACKGROUND: Dendritic cells (DCs) loaded with complex antigen are always used to induce cytotoxic T lymphocytes (CTLs) which have a specific anti-tumor activity. However, CTLs can assault autologous cells induced by DCs loaded with autologous antigen. This study aimed to explore how to weaken the autoimmune reaction induced by DC vaccine by combining mature DC (mDC) activating immunity and immature DC (imDC) leading to immune tolerance to make hepatocellular carcinoma (HCC) vaccine in vitro. METHODS: DC progenitors derived from human peripheral blood were assigned to two groups. One was cultured to mDC and pulsed with frozen-thawed antigen (FTA) of human HCC cell line SMMC-7721 cells (mDC group), and the other was cultured to imDC and pulsed with FTA of human liver cell line L-02 cells (imDC group). The morphology of DCs was monitored and cells phenotypes including HLA-DR, CD80, CD1α, CD83 were assayed by flowcytometry (FCM). The concentrations of interleukin-12 (IL-12) in the supernatant were assayed by ELISA. Methyl thiazolyl tetrazolium (MTT) was used to evaluate T cell proliferation induced by mDC and imDC and the killing rate of CTL induced by mDC and imDC respectively/together on SMMC-7721 and L-02 cells. RESULTS: Compared with the imDC group, the mDC group was characterized by the following: increased secretion of IL-12 (P【0.05); higher expression of HLA-DR, CDla, CD80, CD83; and stronger activity in stimulating proliferation of isogenic T cells (P【0.05). CTL induced by the mDC group had a significant killing response to SMMC-7721 as well as a higher killing rate for L-02 (P】0.05). CTL induced by mDC and imDC together had a higher killing response to SMMC-7721, but a lower killing rate for L-02(P【0.01). CONCLUSIONS: CTL induced by mDC and imDC together has a higher antigen-specific killing response in vitro than that induced by mDC alone. This may be of greater clinical value.

Holotransferrin Enhances Selective Anticancer Activity of Artemisinin against Human Hepatocellular Carcinoma Cells

Artemisinin, also termed qinghaosu, is extracted from the traditional Chinese medicine ar- temesia annua L. （the blue-green herb） in the early 1970s, which has been confirmed for effectively treating malaria, Additionally, emerging data prove that artemisinin exhibits anti-cancer effects against many types of cancers such as leukemia, melanoma, etc. Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill can- cer cells with increased intracellular iron concentrations. This study is aimed to investigate the selective inhibitory effects of artemisinin on SMMC-7721 cells in vitro and determine the effect of holotransfer- fin, which increases the concentration of ferrous iron in cancer cells, combined with artemisinin on the anticancer activity. MTT assay was used for assessing the proliferation of SMMC-7721 cells treated with artemisinin. The induction of apoptosis and inhibition of colony formation in SMMC-7721 cells treated with artemisinin were determined by TdT-mediated dUTP nick end labeling （TUNEL） and col- ony formation assay, respectively. The results showed that artemisinin at various concentrations signifi- cantly inhibited growth, colony formation and cell viability of SMMC-7721 cells （P〈0.05）, likely due to induction of apoptosis of SMMC-7721 cells. Of interest, it was found that incubation of artemisinin combined with holotransferrin sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells （P〈0.01）. Our data suggest that treatment with artemisinin leads to inhibition of viability and pro- liferation, and apoptosis of SMMC-7721 ceils. Furthermore, we observed that holotransferrin signifi- cantly enhanced the anti-cancer activity of artemisinin. This study may provide a potential therapeutic choice for liver cancer.

β-谷甾醇、豆甾醇诱导人肝癌细胞SMMC-7721凋亡

目的研究β-谷甾醇、豆甾醇诱导人肝癌细胞SMMC-7721的凋亡作用。方法 MTT法观察β-谷甾醇、豆甾醇对细胞增殖的抑制作用;激光共聚焦显微镜观察细胞形态变化;用流式细胞仪检测细胞周期、凋亡率、细胞内活性氧ROS、钙离子Ca2+含量、线粒体膜电位ΔΨm变化。结果β-谷甾醇、豆甾醇均明显抑制SMMC-7721细胞增殖,使细胞形态发生典型凋亡变化,凋亡率和细胞内Ca2+、ROS的含量均显著增加,线粒体膜电位降低。β-谷甾醇使细胞周期阻滞在G2/M期,而豆甾醇则同时阻滞在S期和G2/M期。结论β-谷甾醇、豆甾醇具有抑制人肝癌细胞SMMC-7721的增殖和诱导细胞凋亡的作用。

抑制素在人肝癌细胞SMMC-7721和Huh-7中的表达及其编码序列扩增

目的检测抑制素在人肝癌细胞SMMC-7721及Huh-7中的基因表达,扩增抑制素全长编码序列。方法分别提取肝癌细胞SMMC-7721及Huh-7细胞总RNA,逆转录合成cDNA。使用Real time PCR,以GAPDH为内参检测抑制素的mRNA表达情况。采用RT-PCR方法扩增抑制素全长编码序列。结果 Real time PCR结果显示,SMMC-7721和Huh-7肝癌细胞株中均有抑制素的表达,且SMMC-7721细胞中抑制素表达量是Huh-7细胞中的6.652倍。RT-PCR方法扩增得到约835 bp的抑制素全长编码序列,条带特异,无非特异性扩增。结论人肝癌细胞株SMMC-7721中抑制素的表达量较高。

乳香挥发油抑制人肝癌SMMC-7721细胞株增殖及诱导凋亡的作用

目的探讨乳香挥发油体外抑制人肝癌SMMC-7721细胞株增殖及诱导凋亡的作用。方法使用四甲基偶氮唑蓝(MTT)法观察乳香挥发油对SMMC-7721的增殖抑制作用;通过吖啶橙染色,观察SMMC-7721的形态学变化;使用琼脂糖凝胶电泳法检测乳香挥发油对细胞DNA降解的影响;利用流式细胞术分析乳香挥发油诱导SMMC-7721凋亡的凋亡率及对细胞周期的影响;应用间接免疫荧光法观察凋亡调控基因bax和bcl-2蛋白的表达变化。结果乳香挥发油浓度为0.7、0.07、0.007、0.0007、0.00007mg.mL-1作用24h时,能抑制SMMC-7721的生长,并呈浓度依赖性。在浓度为0.07mg.mL-1时,作用48h或72h,电泳结果显示了DNA梯状条带,流式细胞仪检测到凋亡峰,且细胞被阻滞于G1期。间接免疫荧光法的结果表明,促凋亡蛋白bax的表达增强,抗凋亡蛋白bcl-2的表达无明显变化。结论乳香挥发油能抑制肝癌细胞株SMMC-7721的增殖,并可能通过上调线粒体内bax/bcl-2的表达比例诱导SMMC-7721细胞的凋亡,而且其诱导的凋亡具有细胞周期依赖性。

p53途径在氯化两面针碱抑制SMMC-7721细胞增殖中的作用

目的研究氯化两面针碱(nitidine chloride,NC)处理后p53通路的分子水平变化,以说明p53途径在NC抑制人肝癌细胞SMMC-7721增殖中的作用。方法应用MTT比色法测定NC对SMMC-7721细胞存活率的影响,细胞克隆集落形成实验测定NC对细胞增殖的抑制作用;实时荧光定量PCR检测不同剂量NC对p53、p21mRNA表达的影响;酶联免疫吸附法测定p53、p21蛋白含量的变化。结果 NC(0.15,0.30,0.60,1.20,2.40,4.80 mg.L-1)浓度依赖性地降低SMMC-7721的存活率,作用48 h时IC50值为(1.03±0.18)mg.L-1;NC可明显抑制细胞集落形成,剂量为0.15,0.30 mg.L-1时,抑制率分别是74.65%、88.48%,剂量为0.60 mg.L-1时,抑制率达到了100%;NC可明显提高癌细胞中p53、p21 mRNA和蛋白的表达,并呈浓度依赖性。结论 p53通路可能在NC诱导的SMMC-7721细胞增殖抑制中起重要作用。

三氧化二砷对人肝癌SMMC-7721细胞侵袭转移能力的影响

目的:探讨三氧化二砷(As2O3)对人肝癌SMMC-7721细胞黏附、侵袭、转移能力的影响以及其机制。方法:采用MTT法观察人肝癌SMMC-7721细胞经As2O3作用前后对基底膜黏附能力的影响;Transwell膜侵袭系统观察SMMC-7721细胞经As2O3作用前后游走与穿透基底膜能力的改变。免疫细胞化学方法检测人肝癌细胞经As2O3作用前后CD44V6表达的改变。结果:As2O3能显著抑制人肝癌SMMC-7721细胞与m arigel的黏附、抑制细胞游走与穿透基底膜的作用(P<0.05),能抑制人肝癌细胞CD44v6的表达(P<0.05)。结论:As2O3具有抑制人肝癌细胞的侵袭、转移能力,其抑制作用可能与CD44v6的表达下调有关。

陡脉冲电场对体外人肝癌细胞SMMC-7721的诱导凋亡作用

为研究陡脉冲电场的诱导凋亡作用,以体外人肝癌细胞SMMC-7721为实验对象,采用Annexin V-FITC/PI双染色并利用流式细胞术检测磷脂酰丝氨酸(PS)外翻,采用琼脂糖凝胶电泳检测DNA降解片段化。实验发现,陡脉冲电场能有效(P<0.05)地使PS外翻,并且微秒级陡脉冲(μsSPEF)比纳秒级陡脉冲(nsSPEF)更有效(P<0.05);陡脉冲电场能使DNA降解片段化,且nsSPEF比μsSPEF更有效。实验结果表明,陡脉冲电场能有效诱导体外人肝癌细胞凋亡,其机理与陡脉冲电场非热效应及其频谱特性有关。μsSPEF主要作用于细胞膜,而可能通过外源性通路诱导细胞凋亡;nsSPEF主要作用于细胞核和线粒体等胞内细胞器,可能通过内源性通路诱导细胞凋亡。实验结果为陡脉冲电场杀伤肿瘤细胞的机制和参数选择提供了依据。

NF-kB在肝癌细胞株SMMC-7721中的表达及意义

目的 了解NF -kB和ⅠkB在肝癌细胞中的表达情况及意义。方法 通过RT -PCR和Westernblot方法研究NF -kB和ⅠkB在肝癌细胞和正常肝细胞中的mRNA和蛋白表达的变化情况。结果 在肝癌细胞株SMMC -772 1中P65、P5 0mRNA表达增强而ⅠkBmRNA表达下降 (P <0 0 1) ;Westernblot结果显示肝癌细胞株SMMC -772 1细胞的胞核中P5 0、P65蛋白高水平表达 ,而正常肝细胞仅检测出极低水平的P5 0、P65蛋白 ,但是在SMMC -772 1细胞的胞质中ⅠkB蛋白低水平表达 ,正常肝细胞高水平表达。结论 肝癌细胞株SMMC -772 1中NF -kB的P5 0、P65亚基表达升高、ⅠkB表达下降与肿瘤细胞的生长密切相关。

陕重楼甾体皂苷类单体豆甾醇-3-O-β-D-吡喃葡萄糖苷对SMMC-7721细胞增殖的影响

目的:探讨陕重楼甾体皂苷类单体A(BM-26)豆甾醇-3-O-β-D-吡喃葡萄糖苷对人肝癌SMMC-7721细胞增殖的影响。方法:将不同浓度单体A(BM-26)与人肝癌SMMC-7721细胞分别在24、48、72 h处理后,采用MTT法,测定吸光度A值,并计算细胞增殖抑制率,检测陕重楼甾体皂苷类单体A(BM-26)对肿瘤细胞增殖的影响。结果:不同质量浓度陕重楼甾体皂苷类单体A(BM-26)对人肝癌SMMC-7721细胞均有抑制作用:24 h处理时,质量浓度为8、40、200μg·m L^(-1),1、5、25 mg·m L^(-1)组与对照组相比较,吸光度A值有显著性降低(P<0.01或P<0.05);48 h处理时,质量浓度为0.3、1.6、8、40、200μg·m L^(-1),1 mg·m L^(-1)组与对照组相比较,吸光度A值有显著性降低(P<0.01或P<0.05);72 h处理时,质量浓度为8、40μg·m L^(-1)组与对照组相比较,吸光度A值有显著性降低(P<0.01或P<0.05)。其中质量浓度为8μg·m L^(-1)时,在24、48、72 h抑制率分别为45%、41%、36%。结论:陕重楼单体A(BM-26)对人肝癌SMMC-7721细胞增殖具有一定的抑制作用,且最强抑制质量浓度为8μg·m L^(-1),有效抑制质量浓度范围是8~40μg·m L^(-1)。

芪术方诱导人肝癌细胞SMMC-7721凋亡机制研究

目的:探讨芪术方(QZP)诱导人肝癌细胞SMMC-7721凋亡的分子机制。方法:以不同浓度的QZP干预人肝癌细胞SMMC-7721,流式细胞仪检测细胞凋亡及周期分布情况,RT-PCR法检测AKT1、AKT2、Caspase-9mRNA的表达;蛋白质印迹法检测p-AKT、PTEN蛋白表达。结果:QZP能阻滞人肝癌细胞SMMC-7721从G0/G1期进入S期,并诱发细胞凋亡;能抑制AKT1、AKT2表达,提高Caspase-9表达,且跟浓度呈正比例关系。结论:QZP能诱导人肝癌细胞SMMC-7721凋亡和阻滞细胞周期,其机制可能为促进人肝癌细胞SMMC-7721 PTEN表达的增加,使得PI3K/AKT信号通路激活受阻,同时抑制p-AKT、AKT1、AKT2的表达,激活下游Caspase-9促凋亡分子的表达,从而为PI3K/AKT信号通路介导细胞凋亡这一设想提供了理论依据。

SMMC-7721与Bel-7402人肝癌细胞株生长激素受体的表达及意义

目的了解生长激素受体(GHR)在SMMC-7721与Bel-7402人肝癌细胞株的表达情况,以获得肝癌GHR在细胞水平上的定量表达。方法分别采用放射性配体分析与逆转录-多聚酶链式反应(RT-PCR法)检测GHR在SMMC-7721与Bel-7402肝癌细胞株的表达情况,使用双脱氧末端终止法进行基因测序。结果SMMC-7721与Bel-7402肝癌细胞均可表达GHRmRNA;在蛋白质表达水平发现GHR在7721肝癌细胞的位点数量(Site,103/cell)为3.462±0.632,平衡解离常数(Kd)为0.52±0.05942nmol/L;7402肝癌细胞位点数量为7.348±0.891,Kd为0.63±0.04583nmol/L。结论由于上述两种肝癌细胞株均可表达GHR,因此可为研究rhGH与肝细胞肝癌的生长关系奠定一定的实验基础。

姜黄素通过内质网应激信号通路诱导肝癌SMMC-7721细胞凋亡的作用及对细胞活性影响分析

目的分析姜黄素通过内质网应激信号通路诱导肝癌SMMC-7721细胞凋亡的作用及对细胞活性影响。方法选取2017年4月~2018年3月期间为研究时间段,以此期间购置的肝癌SMMC-7721细胞为试验样本,利用不同浓度姜黄素处理试验样本,先经流式细胞仪检测细胞凋亡情况,后经CCK8法分别在试验开始后24 h检测其细胞活力,再经流式细胞仪检测肝癌细胞ROS含量。结果姜黄素浓度越高,肝癌细胞存活率呈逐步下降趋势,同空白对照样本比较,有显著性差异(P<0.05);姜黄素浓度越高,MFI值呈逐步升高趋势,同空白对照样本比较,有显著性差异(P<0.05);不同浓度姜黄素处理的试验样本细胞凋亡率,呈逐渐上升趋势,与空白对照样本比较,有显著性差异(P<0.05)。结论利用姜黄素可有效地抑制肝癌细胞活性,诱导肝癌细胞凋亡。

氯化镉处理人肝癌SMMC-7721细胞株某些生化指标的变化

目的观察氯化镉（CdCl2）处理人肝癌SMMC-7721细胞株某些生化指标的变化。方法利用四甲基偶氮唑蓝（MTY）法检测细胞存活率；利用生化法检测CdCl2处理人肝癌SMMC-7721细胞株丙二醛（MDA）含量和超氧化物歧化酶（SOD）及谷胱甘肽过氧化物酶（GSH．px）活力。结果不同浓度的氯化镉作用J2、24和48h，随着给予CdCl2剂量的增加，细胞存活率显著下降。随着给药时间的延长，对细胞抑制作用增强，存在着剂量．效应关系、时间-效应关系；给予细胞10～40gmol／lCdCl2作用24h，随着给镉剂量增加，细胞的MDA含量增加，SOD和GSH—px的活力下降。结论CdCl2可致人肝癌SMMC-7721细胞株存活率下降和某些氧化和抗氧化生化指标改变有关。

桂皮酸联合Ndfip1对人肝癌细胞SMMC-7721细胞增殖与凋亡的协同效应

目的通过分别检测不同浓度不同作用时间的桂皮酸联合Ndfip1蛋白对人肝癌SMMC-7 721细胞增殖及凋亡作用的影响,阐述中药桂皮酸与Ndfip1蛋白对该肿瘤细胞的作用。方法采用MTT法检测不同浓度不同作用时间的桂皮酸单独及联合Ndfip1对人肝癌细胞SMMC-7 721增殖作用的影响;采用流式细胞术检测不同浓度不同作用时间的桂皮酸单独及联合Ndfip1对人肝癌细胞SMMC-7 721凋亡作用的影响。结果不同浓度的桂皮酸单独及联合Ndfip1作用于人肝癌SMMC-7 721细胞均可造成对细胞增殖的抑制及对细胞凋亡的促进,且联合作用优于单独作用,2者相比具有显著性差异。结论桂皮酸联合Ndfip1蛋白作用时,对人肝癌SMMC-7 721细胞增殖的抑制及对凋亡的促进均较桂皮酸单独作用有明显增强,表明2者的作用可能具有协同效应,这为进一步探索桂皮酸联合Ndfip1蛋白对肝癌细胞的作用奠定了基础。

陕重楼甾体皂苷类单体B(BM-61)对SMMC-7721细胞增殖的影响

目的:观察陕重楼甾体皂苷单体B(BM-61)对人肝癌SMMC-7721细胞的影响。方法:将不同浓度BM-61与人体肝癌SMMC-7721细胞分别在24 h、48 h、72 h处理后,采用MTT法,测定吸光度OD值,并计算细胞增殖抑制率,检测陕重楼单体B对肿瘤细胞增殖的影响。结果:本品1.6 mg·L-1、8 mg·L-1、40 mg·L-1、200 mg·L-1、1 000 mg·L-1剂量组在24 h、48 h、72 h与对照组比较吸光度A值有显著性降低(P<0.01或P<0.05),本品0.3 mg·L-1剂量在24 h、48 h吸光度A值也有显著性降低(P<0.05),其中8 mg·L-1在24 h、48 h、72 h吸光度最小,抑制率分别为19%、23%、17%。结论:陕重楼BM-61对人肝癌SMMC-7721细胞具有一定的抑制作用,且最强抑制浓度为8 mg·L-1。

Hsa-miR-4282对肝癌细胞系SMMC-7721生长的影响

目的探讨Hsa-miR-4282在肝癌细胞系SMMC-7721中的表达及其对细胞生长的影响。方法采用实时荧光定量PCR法检测Hsa-miR-4282在人正常肝上皮细胞系HL-7702和人肝癌细胞系MHCC97-H、SMMC-7721,以及 20例肝癌组织及其相应癌旁组织中的表达。采用瞬时转染法将Hsa-miR-4282 mimics(上调组)和Hsa-miR-4282 inhibitor(下调组)分别转染肝癌SMMC-7721细胞,上调组和下调组分别设置相应阴性对照组。转染后采用MTT法检测细胞增殖能力,平板克隆形成实验检测细胞克隆形成能力,流式细胞仪检测细胞凋亡能力。结果 Hsa-miR-4282在肝癌组织、肝癌细胞MHCC97-H及肝癌细胞SMMC-7721中的表达均低于癌旁组织及正常肝细胞HL-7702(P<0.05)。MTT实验结果显示,Hsa-miR-4282上调后肝癌SMMC-7721细胞的OD值低于其阴性对照组,而下调后OD值高于其阴性对照组(P<0.05)。平板克隆形成实验显示,下调组的细胞克隆数高于其阴性对照组[(240±7)个 vs (191±10)个,P=0.005)],而上调组细胞克隆数低于基阴性对照组[(146±10)个 vs (193±12)个,P=0.013)]。流式细胞仪检测结果显示,Hsa-miR-4282上调组细胞凋亡率较其阴性对照组升高[(23.89±1.89)% vs(16.6±1.14)%,P=0.009)],下调组细胞凋亡率较期阴性对照组降低[(14.98±0.46)% vs (17.79±0.73)%,P=0.010]。结论 Hsa-miR-4282上调可抑制肝癌SMMC-7721细胞增殖,促进细胞凋亡,可能与肝癌的发病机制有关。