Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

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.

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.

Differential Proteomics in Malignant and Normal Liver Cell Lines

Objective： To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods： Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 （Immobilized Mental Affinity Capture） and WCX2 （Weak Cation Exchange） chips on the SELDI-TOF-MS ProteinChip reader. Results： Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion： Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

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。

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。

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。

生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对抗肿瘤药物敏感性的研究

背景:生存素是凋亡抑制蛋白家族的重要成员,生存素基因有可能成为肿瘤反义基因治疗的理想靶基因。目的:研究生存素反义核酸诱导肝癌细胞株SMMC-7721凋亡和增加其对常用抗肿瘤药物敏感性的作用,探讨生存素反义核酸用于肿瘤基因治疗的可能性。方法:应用基因重组技术构建pEGFP-C1-生存素反义核酸重组质粒,以脂质体转染法转染SMMC-7721细胞,用逆转录聚合酶链反应(RT-PCR)检测生存素mRNA的表达,用流式细胞仪检测细胞凋亡。将生存素反义核酸分别与7种常用抗肿瘤药物共同作用于SMMC-7721细胞,用四唑蓝(MTT)比色法测定细胞杀伤率。结果:生存素反义核酸可抑制SMMC-7721细胞中生存素mRNA的表达,从而导致细胞凋亡增加,其作用呈剂量依赖性。生存素反义核酸可增加SMMC-7721细胞对7种常用抗肿瘤药物的敏感性,明显增强药物的杀伤作用。结论:生存素反义核酸能靶向抑制野生型生存素基因的表达,提高肝癌细胞对常用抗肿瘤药物的敏感性,有可能成为肿瘤基因治疗的新方法。

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。

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)。

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。

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。

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。

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。

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。

siRNA沉默Cyclin E基因对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响

目的:观察siRNA沉默Cyclin E基因表达对肝癌HepG2、SMMC-7721和BEL-7402细胞增殖和侵袭能力的影响.方法:构建2个靶向Cyclin E基因siRNA载体,转染人肝癌HepG2、SMMC-7721和BEL-7402细胞.RT-PCR、Western blot检测转染后HepG2、SMMC-7721和BEL-7402细胞Cyclin E基因mRNA和蛋白表达水平.CCK-8试验、软琼脂克隆形成实验检测HepG2、SMMC-7721和BEL-7402细胞增殖、克隆形成能力.流式细胞术、transwell试验分别检测HepG2、SMMC-7721和BEL-7402细胞周期和侵袭能力.结果:构建的2个Cyclin E基因siRNA载体插入序列与所设计序列均一致;转染HepG2、SMMC-7721和BEL-7402细胞后,干扰1组、干扰2组与空白对照组和阴性对照组比较,C y c l i n E m R N A和蛋白表达量均显著降低(P<0.05),细胞生长速度延缓,软琼脂细胞集落形成数、穿透细胞数均显著降低(P<0.05),S和G2/M期细胞比例减少,G0/G1期细胞比例增加.结论:沉默肝癌细胞Cyclin E表达水平,可有效抑制细胞生长、增殖和侵袭能力.