肝泰煎剂含药血清诱导人肝癌细胞系Bel-7402细胞的凋亡

目的:观察肝泰煎剂(GTJJ)含药血清诱导人肝癌细胞系Bel-7402细胞凋亡现象。方法:将GTJJ含药血清作用于人肝癌细胞系Bel-7402细胞,应用MTT检测对肝癌细胞生长抑制作用,倒置显微镜、荧光显微镜、激光共聚焦扫描显微镜等影像学方法观察细胞形态学变化,以及PI染色单染、AnnexinV-PI双染后,流式细胞术检测对细胞周期影响及诱导细胞凋亡程度。结果:MTT法检测结果显示:GTJJ含药血清具有抑制Bel-7402肿瘤细胞生长作用[其中20%GTJJ等效剂量抑制率50.09%,与生理盐水(NS)组比,P<0.01];荧光显微镜及激光共聚焦显微镜可观察到典型的凋亡形态学变化;流式细胞术检测显示,细胞周期出现G0/G1期阻滞,并出现典型的凋亡峰;AnnexinV-PI双染法检测到早期及中晚期细胞凋亡。结论:GTJJ含药血清有诱导细胞凋亡作用。     

消瘤汤含药血清对人肝癌细胞Bel-7402增殖和凋亡的影响

[目的]观察消瘤汤含药血清对人肝癌细胞Bel-7402增殖和凋亡的影响。[方法]采用消瘤汤含药血清,应用血清药理学方法,选择不同浓度的含药血清体外培养人肝癌细胞Bel-7402 96 h。通过MTT法和细胞形态学观察检测细胞的增殖抑制率和细胞凋亡。[结果]MTT法检测表明,消瘤汤高、中、低剂量组与0.95%氯化钠（对照）组相比,对人肝癌细胞增殖均有抑制作用（P〈0.01）。细胞形态学检测表明消瘤汤各剂量组含药血清均可诱导细胞凋亡。[结论]消瘤汤能明显抑制肝癌Bel-7402细胞的增殖,其抑瘤作用有细胞毒作用和诱导细胞凋亡2种途径。     

剔毒护肝颗粒含药血清对人肝癌细胞增殖与凋亡的影响

目的:探讨中药剔毒护肝颗粒剂(TDHGG)含药血清对人肝癌bel-7402细胞生长及凋亡的影响.方法:应用MTT比色法检测TDHGG对肝癌细胞的抑制作用;应用流式细胞仪检测细胞凋亡率.结果:肿瘤细胞抑制率与药物剂量呈直线相关;流式细胞仪检测结果显示含药血清大、中、小剂量处理组,肝癌细胞的凋亡率分别为23.9%、18.1%、5.8%,明显高于对照组(P＜0.05),其中G0/G1期细胞数增加,G2/M期细胞数明显减少,与对照组相比差异有显著性意义(P＜0.01).结论:TDHGG含药血清可抑制人肝癌bel-7402细胞的生长,并可干扰肝癌细胞增殖周期,诱导肝癌细胞凋亡.     

As2O3联合Aspirin对诱导肝癌细胞凋亡的影响

目的:观察As2O3与Aspirin联合应用对肝癌细胞Bel-7402的影响,并探讨其作用机制.方法:体外培养肝癌Bel-7402细胞,Aspirin、As2O3不同浓度孵育细胞.倒置显微镜观察细胞形态学改变,四甲基偶氮唑蓝(MTT)法检测As2O3和Aspirin单独及联合应用对Bel-7402细胞增殖情况的影响,流式细胞术观察细胞凋亡情况,并通过流式软件分析细胞周期变化.结果:As2O3及Aspirin对肝癌Bel-7402细胞生长均呈不同程度的抑制,且呈浓度依赖性.二者联合具有协同作用,药物联用组抑制率均显著高于单独应用同等剂量As2O3组和Aspirin组(P<0.05).2.0μmol/LAs2O3与0.2mmol/LAspirin联用组与2.0μmol/LAs2O3单药组相比,凋亡率从5.64%±0.56%提高到7.35%±0.62%,差异具有统计学意义(P<0.05).且通过对两组细胞周期检测发现,G1期细胞从40.52%±0.64%下降到32.03%±0.97%,G2期及S期细胞分别从9.57%±0.82%、50.41%±0.32%上升到13.66%±0.82%、54.37%±0.69%,Aspirin能显著增强As2O3诱导细胞集中于S及G2期的作用,从而增强其促凋亡作用.结论:Aspirin能增强As2O3诱导细胞凋亡的作用,该作用可能与影响细胞周期有关.     

含蟾酥胶囊血清诱导人肝癌细胞株凋亡及机制探讨

目的观察含蟾酥胶囊（CC）血清诱导人肝癌细胞株BEL-7402凋亡作用,并探讨其机制。方法体外培养的BEL-7402加入含不同浓度CC血清,分别孵育24、48h,显微镜下观察细胞形态;MTT比色测算细胞抑制率;流式细胞术测算细胞凋亡率;琼脂糖凝胶电泳测定其DNA梯状条带;免疫细胞化学染色检测细胞中Bcl-2表达变化。结果与不加CC血清比较,加入含CC血清的BEL-7402呈凋亡细胞形态学改变;细胞生长抑制率、凋亡率升高;孵育48h时,琼脂糖凝胶电泳呈现DNA梯状条带;细胞Bcl-2表达明显降低。结论CC可以诱导BEL-7402凋亡,其作用机制可能与下调细胞Bcl-2表达有关。     

892号药液血清诱导人肝癌细胞凋亡的初步研究

目的:探讨中药复方892号药波对人肝癌细胞株(Bel-7402)凋亡的诱导作用。方法:以血清药理学方法,选择不同浓度的含药血清与人肝癌细胞共同孵育不同时间后,应用倒置相差显微镜、荧光显微镜对细胞生长状况和荧光染色后的细胞核形态进行观察。结果:人肝癌细胞经含中药血清孵育处理后,由贴壁生长而逐渐脱落,外形变圆,体积缩小,折光性增强;荧光镜下见细胞核破碎,裂解为大小不等的凋亡小体。结论:中药复方892号药液可诱导人肝癌细胞的凋亡,达到抑制肿瘤细胞生长的目的。     

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.     

PEG10基因RNA干扰对人肝癌细胞系Hep3B体外增殖的影响

目的研究PEG10基因RNA干扰对肝癌细胞系Hep3B体外增殖的影响。方法psiRNA-PEG10真核表达载体转染Hep3B细胞,MTT法检测细胞体外增殖速度,流式细胞仪检测细胞周期变化。结果psiRNA-PEG10真核表达载体转染Hep3B细胞后,明显沉默了Hep3B mRNA和蛋白的表达,转染后48 h抑制效率最高。Hep3B细胞体外增殖受到抑制（P〈0.05）,流式细胞仪分析结果显示大量Hep3B细胞凋亡,激光共聚焦显微镜观察到凋亡细胞形态。结论PEG10基因RNA干扰在体外可明显沉默肝癌细胞内PEG10基因的表达,抑制肝癌细胞的生长,诱导细胞凋亡。     

肝癓口服液含药血清对转化生长因子α诱导人肝癌细胞SMMC-7721增殖和凋亡的研究

目的 :观察肝疒徵 口服液含药血清对转化生长因子 (TGFα)诱导人肝癌细胞增殖和凋亡的影响。方法 :采用血清药理学方法 ,以病理鼠血清作对照 ,应用MTT比色法观察中药鼠血清对TGFα诱导人肝癌细胞SMMC 772 1增殖的抑制作用。用流式细胞术检测中药血清对肝癌细胞凋亡和细胞周期的影响。结果 :中药血清对TGFα诱导的肝癌细胞SMMC 772 1增殖有明显的抑制作用 ,抑制效应呈时间依赖性。中药血清能促进肝癌细胞凋亡 ,使细胞滞留于G0 1 期 ,降低S期细胞百分比和增殖指数。结论 :肝疒徵 口服液含药血清能够抑制TGFα诱导的人肝癌细胞增殖 ,促进其凋亡。     

益气活血软坚解毒方含药血清诱导人肝癌细胞系Bel-7402细胞凋亡过程中部分凋亡调控基因变化

目的:研究益气活血软坚解毒方(YHRJ)含药血清对人肝癌细胞系Bel-7402细胞凋亡调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kB表达影响.方法:将细胞分为对照组(NS)组、NS DDP(顺氯氨铂)组、YHRJ等剂量组(YHRJD)、YHRJD DDP组、YHRJ高剂量组(YHRJG)、YHRJG DDP组,应用流式细胞术、免疫组化、原位杂交、RT-PCR等方法对用药24,48h的Bel-7402细胞凋亡的主要调控基因Fas,FasL,Bcl-2,Bax,P53,NF-kBmRNA与蛋白表达进行检测.结果:流式细胞术检测显示:与NS组相比,NS DDP、YHRJD、YHRJD DDP及YHRJG DDP组Fas蛋白表达均明显提高(30.12%±22.94%,10.50%±8.41%,30.35%±22.98%,32.61%±26.87%vs8.77%±6.93%,P<0.01),YHRJG组效果不明显(P>0.05);NS DDP、YHRJD DDP组FasL蛋白表达也升高(16.40%±7.168%,8.41%±6.74%vs4.12%±2.60%,P<0.01),而YHRJG组FasL蛋白表达降低(3.05%±2.53%vs4.12%±2.60%,P<0.01).免疫组化结果显示:除YHRJG DDP组外,其余各组突变型P53蛋白表达明显降低(30.2%,14.6%,19.8%,17.3%vs60.0%,P<0.05);各加药组Bax蛋白表达明显增高(40.7%,40.4%,72.1%,68.9%,42.2%vs30.0%,P<0.05);NS DDP组与YHRJG组Bcl-2蛋白表达明显降低(26.3%,24.4%vs30.5%,P<0.05),而YHRJD、YHRJG DDP、YHRJG DDP组Bcl-2表达明显升高(41.8%,39.3%,45.6%vs30.5%,P<0.05);各加药组NF-kB蛋白表达明显降低(15.9%,13.3%,14.1%,7.8%,14.6%vs24.2%,P<0.05).原位杂交结果显示:各加药组NF-kBmRNA表达明显降低(30.5%,13.3%,21.4%,17.4%,53.2%vs58%,P<0.05).RT-PCR结果显示:YHRJ等效剂组凋亡调控基因Bcl-2mRNA表达明显降低(0.717±0.198vs1.327±0.097,P<0.001);DDP、YHRJD、YHRJG组凋亡调控基因BaxmRNA表达明显增高(46.22±6.22,56.19±7.36,62.32±11.06vs35.22±4.38,P<0.05).结论:YHRJ含药血清诱导人肝癌细胞系Bel-7402细胞凋亡可能的基因调控机制在于通过抑制凋亡信号转导基因FasL基因蛋白表达,促进凋亡调控基因Bax基因蛋白表达,抑制NF-kB基因mRNA及蛋白表达来实现的.     

Melatonin and Doxorubicin synergistically induce cell apoptosis in human hepatoma cell lines

AIM:To investigate whether Melatonin has synergistic effects with Doxorubicin in the growth-inhibition and apoptosis-induction of human hepatoma cell lines HepG2 and Bel-7402.METHODS:The synergism of Melatonin and Doxorubicin inhibited the cell growth and induced cell apoptosis in human hepatoma cell lines HepG2 and Bel-7402.Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay.Cell apoptosis was evaluated using TUNEL method and flow cytometry.Apoptosis-r...     

Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis

AIM: To investigate the cyclooxygenase-2 (COX-2)expression level in human HepG2, Bel-7402 and SMMC-7721hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis.METHODS: Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium (MTT) bromide colorimetric assay. Celecoxibinduced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis.RESULTS: Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTr assays and morphological changes.The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%,and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3and dephosphorylation of Akt (Thr308). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr308) after treatment with celecoxib.CONCLUSION: COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.     

Overexpression of cyclooxygenase-2 in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and mechanism of cyclooxygenase-2 selective inhibitor celecoxib-induced cell growth inhibition and apoptosis

AIM: To investigate the cyclooxygenase-2 (COX-2) expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis. METHODS: Hepatoma cells were cultured and treated with celecoxib. Cell In situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenylte-trazolium (MTT) bromide colorimetric assay. Celecoxib-induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis. RESULTS: Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%, and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt (Thr308). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr308) after treatment with celecoxib. CONCLUSION: COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.     

Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis

AIM: To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma (HCC) cells and to explore the underlying mechanism. METHODS: Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber. p125FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice. Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Microvessels with immunohistochemical staining were detected. RESULTS: Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11-28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125FAK in 5 μg/mL genistein group (15.26±0.16%) and 10 μg/mL genistein group (12.89±0.36%) was significantly lower than that in the control group (19.75±1.12%,P<0.05). Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group (10.422±0.807) was significantly lower than that in control group (22.330±5.696, P< 0.01). CONCLUSION: Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.     

Effects of aqueous extracts of Aconitum carmichaeli,Rhizoma bolbostemmatis,Phytolacca acinosa,Panax notoginseng and Gekko swinhonis Gūenther on Bel-7402 cells

AIM: To investigate the anticancer activity of a chinese medical mixture, WRCP (warming and relieving Cold Phlegm), on hepatocarcinoma Bel-7402 cells. METHODS: Fingerprints of WRCP, which were composed of aqueous extracts of Aconitum carmichaeli, Rhizoma bolbostemmatis, Phytolacca acinosa, Panax notoginseng and Gekko swinhonis Gūenther, and aconitine, which could be isolated from Aconitum carmichaeli and have the potential toxicity, were identified by high pressure liquid chromatography. Bel-7402 cells were grown in the presence of WRCP, As2O3 or all-trans-retinoic acid (ATRA). Cell proliferation and viability were determined by trypan blue stain. Apoptosis and cell cycle of Bel-7402 cells were detected by flow cytometry. Morphologic and ultrastructural variations were determined under optic and electronic microscopy. The secretion of alpha-fetoprotein and albumin was detected by radioimmunoassay. RESULTS: The average quality of aconitine is 1.15 ± 0.10 μg per 7.5 g extracts. WRCP could suppress the proliferation and viability of Bel-7402 cells. The percentage of apoptosis cells and S phase cells increased on WRCP-treated cells. Treated with WRCP, Bel-7402 cells showed ultrastructural features of differentiation. The alpha-fetoprotein secretion decreased while the albumin secretion increased (P < 0.001, P < 0.001, respectively) markedly in WRCP-treated cells.CONCLUSION: WRCP can affect the proliferation, differentiation and apoptosis of Bel-7402 cells. It can arrest cells in S phase and has strong cytotoxicity to Bel-7402 cells.     

反义局部粘着斑激酶脱氧寡核苷酸对肝癌细胞侵袭性生长的抑制作用

目的 观察反义局部粘着斑激酶脱氧寡核苷酸(FAK ODN)转染对肝癌细胞侵袭性生长的影响,并探讨其作用机制。 方法 以LipofecTAMINE介导的反义FAK ODN转染Bel 7402肝癌细胞株,测定Bel 7402肝癌细胞株体外生长曲线、细胞活力,测定不同时间点该细胞体外黏附能力变化,以Transwell小室测定细胞的体外侵袭能力,同时行FAK表达与细胞DNA含量的双参数流式细胞仪检测及细胞凋亡的流式细胞仪检测。 结果 p125FAK表达在反义转染组(6.49％±0.10％)显著低于正义转染组(14.33％±1.88％)与对照组(16.68％±1.62％),F=7.66,P<0.01;反义FAK ODN转染显著抑制Bel 7402肝癌细胞株的生长,其细胞活力显著下降,肿瘤细胞抑制率在30％-60％之间;细胞体外黏附能力受到显著抑制,黏附抑制率在25％-55％间;细胞的体外侵袭能力显著下降,侵袭抑制率在15％-25％之间;细胞凋亡显著增加;细胞周期分析显示S期细胞比率显著降低,细胞生长主要阻滞在G2/M期。 结论 FAK在Bel 7402肝癌细胞的黏附与迁移运动中发挥重要作用,其表达阻断显著抑制肝癌细胞的体外黏附与侵袭活性。FAK表达阻断显著抑制肝癌细胞的体外增殖,促进细胞凋亡。     

靶向甲硫氨酸腺苷转移酶2A基因小干扰RNA抑制肝癌细胞生长的研究

目的研究靶向甲硫氨酸腺苷转移酶(MAT)2A基因的小干扰RNA(siRNA)对肝癌细胞生长和细胞凋亡的影响。方法以MAT 2A为目的基因,以产生siRNA质粒载体pSilence-2.1-U6为表达模板,细胞内转录合成4条siRNA;并构建携带荧光素酶报告基因的重组质粒载体plucA-MAT 2A。脂质体转染法将重组质粒载体plucA-MAT 2A与产生siRNA的质粒pSilence-2.1-U6共转染293 T细胞,定量检测荧光素酶活性,初步筛选出抑制荧光素酶表达的有效siRNA,然后将有效的siRNA转染Bel-7402肝癌细胞,半定量逆转录聚合酶链反应检测MAT 2A mRNA表达,并检测转染后肝癌细胞MAT的活性,进一步采用四甲基偶氮唑盐法观察siRNA对肝癌细胞生长的抑制率,用流式细胞仪检测siRNA对肝癌细胞凋亡的影响。结果所合成的4条siRNA中有2条抑制荧光素酶表达,抑制效率分别为81%和89%,并特异性抑制肝癌细胞MAT 2A表达,降低了肝癌细胞中MAT活性,抑制肝癌细胞的生长,诱导肝癌细胞凋亡。结论靶向MAT 2A基因的siRNA抑制肝癌细胞生长,诱导肝癌细胞凋亡;MAT 2A是肝癌基因治疗的一个很有希望的靶位点。     

犀黄丸诱导Bel-7402细胞凋亡及其细胞内钙离子浓度的变化

研究犀黄丸(含药血清)诱导肝癌Bel—7402细胞凋亡的作用及凋亡过程中细胞内游离钙离子浓度的变化。DNA琼脂糖凝胶电泳法检测细胞凋亡，流式细胞仪检测凋亡过程中细胞内游离钙离子浓度的变化。DNA琼脂糖凝胶电泳法显示有明显的DNA梯带，同时在细胞凋亡过程中细胞内游离钙离子浓度明显升高，以凋亡早期较为明显。犀黄丸能诱导肝癌细胞凋亡，细胞内游离钙离子浓度升高可能是其机制之一。