四藤方对Bel-7402细胞增殖和凋亡作用研究

目的:观察清热解毒中药-四藤方对肝癌细胞Bel-7402增殖和凋亡的影响。方法:不同浓度四藤方处理人肝癌细胞Bel-7402、人肝细胞HL-7702,WST-8法检测细胞增殖,流式细胞术检测细胞凋亡,Hoechst 33258染色检测细胞凋亡形态变化,Western Blot法检测半胱天冬酶-3(caspase-3)活化。结果:与对照组比较,四藤方对人肝细胞HL-7702增殖无明显影响;100～400μg.mL-1四藤方作用24 h可显著抑制肝癌Bel-7402细胞增殖,促使细胞Bel-7402凋亡,呈现细胞凋亡形态,并可活化Bel-7402细胞caspase-3。结论:四藤方可以抑制Bel-7402细胞增殖,促Bel-7402细胞凋亡,并可能与活化caspase-3相关。     

绞股蓝总皂苷对小鼠白血病L1210细胞抑制作用初探

目的:探讨绞股蓝总皂苷(Gypenosides,Gyp)对小鼠白血病L1210细胞生长抑制的作用机制。方法:采用MTT法检测Gyp对体外培养的小鼠L1210细胞的增殖抑制作用,流式细胞仪检测Gyp对L1210细胞线粒体膜电位的影响;DAPI染色检测Gyp对L1210白血病细胞核的变化;单细胞凝胶电泳法检测Gyp对白血病L1210细胞DNA损伤的影响。结果:浓度为100～500μg/mL的Gyp对L1210细胞增殖生长有抑制作用,350μg/mL的Gyp作用于L1210细胞0、12、24 h,线粒体膜电位显著下降;Gyp(350μg/mL)作用4 h时,细胞的活性氧产量最高。Gyp(350μg/mL)作用4、12、24 h后,单细胞凝胶电泳观测到DNA损伤;DAPI染色发现细胞核发生变化,并有时间依赖性。结论:Gyp能抑制L1210细胞增殖,这种抑制作用与活性氧的产生、线粒体膜电位下降和DNA损伤有关。     

华蟾素对人肝癌细胞Bel-7402细胞增殖及周期的影响

目的:观察华蟾素注射液对人肝癌Bel-7402细胞增殖及周期的影响.方法:将不同浓度的华蟾素注射液作用于Bel-7402细胞,应用噻唑蓝还原法(MTT比色法)检测华蟾素注射液对Bel-7402细胞增殖的影响;应用流式细胞术(FCM)检测Bel-7402细胞周期分布.结果:华蟾素注射液可以抑制Bel-7402细胞增殖,其抑制作用与作用时间和剂量呈正相关,并可使细胞阻滞于G2/M期.结论:华蟾素注射液能够抑制人肝癌细胞Bel-7402的增殖,并可使细胞阻滞于G2/M期,诱导细胞凋亡.     

枸杞多糖对人肝癌细胞Bel-7402的抑制作用及凋亡的研究

目的 研究枸杞多糖诱导人肝癌细胞Bel-7402的凋亡作用及其作用机制.方法 体外培养的人肝癌细胞Bel-7402,细胞经不同剂量枸杞多糖(LBP)处理后,采用MTT法测定细胞的增殖情况,流式细胞仪分析LBP对Bel-7402细胞周期和凋亡的影响,用RT-PCR技术检测Bel-7402细胞SurvivinmRNA的表达.结果 LBP可明显抑制人肝癌细胞Bel-7402的生长,并能诱导Bel-7402细胞凋亡,凋亡率(%)分别为(6.20±0.62),(12.80±0.47)和(18.10±0.70),与对照组比较,差异有非常显著性(P<0.01);各剂量药物处理组的肿瘤细胞Survivin mRNA表达水平均明显降低(P<0.01).结论 枸杞多糖可抑制Bel-7402细胞增殖,诱导细胞凋亡,该作用可能与下调细胞Survivin 表达有关.     

人参多糖注射液对肝细胞癌的增殖抑制作用及机制探讨

目的:观察人参多糖注射液对人肝癌细胞BEL-7402和HpeG2增殖抑制作用,并探讨其机制。方法:将不同浓度的人参多糖注射液与人肝癌细胞BEL-7402和HpeG2作用,采用cell counting kit-8(CCK8)检测细胞的增殖抑制,通过倒置显微镜观察用药前后细胞形态、数量变化,透射电镜观察细胞坏死、凋亡等形态学变化,蛋白免疫印迹法(Western blot)检测肿瘤坏死因子受体1(TNFR1)蛋白表达情况。结果:人参多糖注射液对人肝癌细胞BEL-7402和HpeG2的增殖有明显抑制作用(P0.05),并且与浓度和时间呈正相关。60,80,100 g·L~(-1)人参多糖注射液作用于人肝癌细胞BEL-7402和HpeG2 24,48,72h后,可显著抑制其增殖(P0.01),40 g·L~(-1)人参多糖注射液作用于人肝癌细胞BEL-7402和HpeG2 24,48,72 h后可明显抑制其增殖(P0.05)。人参多糖注射液作用人肝癌细胞BEL-7402和HpeG2 48 h后,细胞出现细胞核边集、凋亡小体等凋亡形态变化,72 h后部分细胞出现空泡样变等形态学变化。与空白组比较,40,60,80 g·L-1人参多糖注射液组作用人肝癌细胞BEL-7402和HpeG2后,TNFR1蛋白相对表达量明显升高(P0.05,P0.01)。结论:人参多糖注射液对人肝癌细胞BEL-7402和HpeG2的增殖有明显的抑制作用;诱导细胞凋亡可能是抑制作用的机制。     

雷公藤甲素对人肝癌Bel-7402细胞增殖及凋亡的影响

目的:研究雷公藤甲素影响人肝癌Bel-7402细胞增殖及凋亡的作用及机制。方法:采用细胞药理学实验方法,人肝癌细胞株Bel-7402与雷公藤甲素(10μg/mL、5μg/mL及0.25μg/mL)共培养,MTT法检测其对Bel-7402细胞增殖的影响,TUNEL技术检测其对Bel-7402细胞凋亡的影响,免疫组织化学(SABC)染色法检测Bax及Bcl-2蛋白表达。结果:作用48h后雷公藤甲素(10μg/mL、5μg/mL、0.25μg/mL)3个剂量对Bel-7402细胞增殖均有显著抑制作用,P<0.01,抑制作用随药物浓度增高而有加强趋势,最高抑制率可达38.67%,且呈量效关系,3个剂量组间有显著性差异,P<0.01。雷公藤甲素(10μg/mL、5μg/mL、0.25μg/mL)能有效诱导Bel-7402细胞凋亡,作用显著,P<0.01,且呈显著的量效关系。雷公藤甲素(10μg/mL、5μg/mL、0.25μg/mL)用药组Bax、Bcl-2表达与对照组比较,无显著性差异,P>0.05。结论:雷公藤甲素能抑制人肝癌Bel-7402细胞增殖和诱导其凋亡,其诱导Bel-7402细胞凋亡的作用机制可能不是通过Bax、Bcl-2途径。     

麝黄消瘤汤诱导人肝癌细胞系(Bel-7402)凋亡的研究

目的:研究麝黄消瘤汤对人肝癌细胞系(Bel-7402)的诱导凋亡作用.方法:制备含药血清,孵化人肝癌细胞系(Bel-7402),用电子显微镜观察法、荧光显微镜观察法研究凋亡细胞超微结构,用流式细胞仪检测凋亡细胞DNA直方图及凋亡率.结果:电子显微镜及荧光显微镜下,麝黄消瘤汤含药血清诱导人肝癌细胞系(Bel-7402)出现凋亡征象,最大凋亡率为50.96%.流式细胞仪检测凋亡细胞DNA直方图上出现亚二倍体峰,细胞周期分布频率与对照组有显著区别(P≤0.05).结论:麝黄消瘤汤对人肝癌细胞系(Bel-7402)有显著的诱导凋亡作用.     

复合中药多糖对人肝癌Bel-7402细胞株抗癌作用研究

目的：探讨复方中药多糖对人肝癌Bel-7402细胞株的影响。方法：采用T淋巴细胞转化试验观察复合多糖对人肝癌小鼠细胞，屯疫功能的影响；采用MTT法观察复合多糖对Bel-7402株的细胞增殖抑制；采用流式细胞仪观察复合多糖对Bel-7402株细胞凋亡的影响。结果：复合多糖为1．5、0．75、0．25g，kg剂量时可提高Bel-7402小鼠的淋巴细胞转化功能；复合多糖在40、20mg／L浓度时对Bel-7402细胞株增殖具有抑制作用；复合多糖浓度在40、200、10mg／L时作用24、48、72h使Bel-7402株细胞凋亡率增加。结论：复合多糖可提高Bel-7402小鼠的淋巴细胞转化功能；对Bel-7402细胞株增殖具有抑制作用；使Bel-7402株细胞凋亡率增加，并呈现时间、浓度依赖性。     

翻白草诱导人肝癌细胞HepG-2凋亡的研究(英文)

目的:研究翻白草乙醇提取物对人肝癌细胞HepG-2体外增殖的抑制作用及凋亡的诱导作用。方法:用不同浓度的翻白草乙醇提取物对人肝癌细胞HepG-2,MTT法测定对细胞生长的抑制率,用琼脂糖凝胶电泳观察DNA梯状条带和DAPI染色观察凋亡小体,再用Western blot法检测对caspase-3和PARP蛋白表达的影响。结果:翻白草乙醇提取物对人肝癌细胞HepG-2有较强的浓度依赖性抑制作用,诱导细胞出现明显的形态学改变,琼脂糖凝胶电泳出现典型的DNA梯状条带,Western blot分析结果发现,翻白草乙醇提取物促进caspase-3活化而引发下游PARP裂解,进而诱导人肝癌细胞HepG-2细胞走向自体凋亡的途径。结论:翻白草乙醇提取物可能通过抑制人肝癌细胞HepG-2的增殖及诱导其凋亡而产生抗肿瘤作用。     

蒺藜皂苷对人肝癌细胞BEL7402生长抑制和诱导凋亡作用的研究

目的 :探讨蒺藜皂苷 (STT)对体外培养的人肝癌细胞BEL-7402的增殖抑制作用及诱导凋亡作用。方法 :应用MTT与SRB法检测蒺藜皂苷对人肝癌细胞BEL-7402的抑制作用 ,采用形态学观察以及流式细胞术 (FCM)检测了STT诱导人肝癌细胞BEL-7402凋亡的效应 ,采用流式细胞术检测BEL-7402细胞Bcl-2蛋白表达。结果 :①STT能够剂量依赖地抑制人肝癌细胞BEL-7402的增殖 ;②细胞显现典型的凋亡形态学改变 ,FCM出现凋亡峰 ;③STT能降低BEL-7402细胞的Bcl-2蛋白表达。结论 :STT在体外能抑制BEL-7402细胞的增殖并能诱导细胞凋亡 ,其诱导凋亡作用的途径之一是下调Bcl-2蛋白表达。     

Mechanisms of pseudolaric acid B-induced apoptosis in Bel-7402 cell lines

Previous studies have shown that pseudolaric acid B (PB) would cause apoptosis in human tumor cell lines. However, the mechanisms of PB induced apoptosis are still unclear. In the present study, the mechanisms of PB induced apoptosis in the human hepatocellular carcinoma Bel-7402 cell line were investigated by measuring cell viability, rate of apoptosis, cell cycle, detecting DNA fragmentation, and measuring caspase-3 activation. The results indicated that PB inhibited Bel-7402 cell viability and induced cell death by causing DNA fragmentation, up regulating the early and late apoptotic rates, activating caspase-3 protein, and detaining the cell cycle in the G2/M phases. Additionally, PB-induced apoptosis was a dose- and time-dependent manner. These observations suggest that PB-induced apoptosis occurs through a caspase-dependent pathway and detains the cell cycle in the G2/M phase.     

白藜芦醇抗肝癌Bel-7402及调节荷瘤鼠IL-8分泌机制的研究

目的：探讨白藜芦醇离体与在体对肝癌细胞Bel-7402的抑制作用及可能的效应机制。方法：MTT比色法测定Bel-7402细胞生长抑制率,Western blotting测定Bel-7402细胞Bcl-2水平,流式细胞术分析细胞周期,流式细胞染色检测细胞内IL-8,半定量RT-PCR与双抗体夹心ELISA法检测荷瘤小鼠IL-8 mRNA表达与含量。结果：白藜芦醇可以时间-剂量依赖性抑制Bel-7402细胞增殖,诱导凋亡,影响细胞周期分布。此外,白藜芦醇还能剂量依赖性抑制荷瘤小鼠细胞内IL-8产生及其mRNA与蛋白含量。结论：白藜芦醇离体与在体均能发挥有效的抗肝癌生物学活性和免疫调节作用。     

蛇葡萄素的血管生成抑制作用

目的:探讨蛇葡萄素对内皮细胞及肿瘤细胞的生长及其血管生成的影响。方法:体外用MTT法对牛主动脉内皮细胞进行增殖抑制实验;用ELISA法、免疫组化染色法以及流式细胞仪观察蛇葡萄素对人肝癌细胞Bel-7402分泌VEGF、bFGF的抑制作用;体内观察蛇葡萄素对人肝癌Bel-7402裸鼠移植瘤的抑制作用。结果:蛇葡萄素对牛主动脉内皮细胞的增殖在6.4~51.2μg/m l呈现浓度依赖性抑制作用,IC50=22.0±4.0μg/m l。ELISA法测定结果显示,12.8μg/m l、25.6μg/m l、38.4μg/m l蛇葡萄素对肝癌Bel-7402细胞分泌VEGF的抑制率分别为14.2%、40.0%及49.6%。免疫组化染色结果显示,12.8μg/m l以上的蛇葡萄素能使细胞体积缩小,分泌VEGF、bFGF明显减少。流式细胞仪检测VEGF、bFGF结果,25.6μg/m l及38.4μg/m l蛇葡萄素对VGEF的抑制率分别为32.2%及57.4%,对bFGF的抑制率分别为54.9%及62.6%。蛇葡萄素浓度为100、150、200 mg/kg时对人肝癌Bel-7402裸鼠移植瘤的抑制率分别可达24.3%、41.4%及45.7%。结论:蛇葡萄素在体外能有效抑制内皮细胞增殖及人肝癌Bel-7402细胞分泌VEGF和bFGF,具有抑制血管生成的作用;体内能有效抑制人肝癌Bel-7402裸鼠移植瘤的生长,提示蛇葡萄素也许可作为一个肿瘤血管生成抑制剂用于肿瘤防治。     

苦楝皮中5个柠檬苦素化合物的细胞毒活性以及构效关系（英文）

目的：研究柠檬苦素化合物1-5的细胞毒活性以及阐明其构效关系。方法：从苦楝皮中分离获得化合物1-5,采用MTT法进行3种肿瘤细胞株的体外细胞毒活性实验。结果：化合物1-5均表现出潜在的抗肿瘤活性。尤其是化合物1对人肝癌BEL7402细胞的IC508.57μg·mL-1,对人肺癌H460细胞的IC506.29μg·mL-1,对人胃癌SGC-7901细胞的IC506.08μg·mL-1,较其他化合物表现出更强的细胞毒活性。结论：化合物1-5的构效关系研究显示16位羟基为柠檬苦素化合物具备细胞毒活性的重要基团。     

白桦脂酸抑制食管鳞状细胞癌增殖及相关机制的实验研究

 目的研究抗肿瘤药物白桦脂酸（BA）对人食管鳞癌KYSE170细胞的抑制作用及抗肿瘤作用机制。方法采用不同质量浓度(0、5、10、20、40、60、80、100 μg·mL-1)白桦脂酸处理KYSE170细胞,作用不同时间 (24、48、72 h)后 ,噻唑蓝（MTT）法检测KYSE170 细胞生长的短期抑制作用;不同质量浓度（0、5、20、40 μg·mL-1）白桦脂酸作用于细胞24 h后以克隆形成实验检测药物对细胞生长的长期抑制作用;不同质量浓度白桦脂酸 (10、60、100 μg·mL-1)作用KYSE170细胞 24、48 h后以流式细胞仪FITC Annexin V/PI双染法检测细胞凋亡变化,不同质量浓度白桦脂酸（0、10、40 μg·mL-1）作用细胞24 h后以流式细胞仪检测细胞周期的改变。结果噻唑蓝实验显示,不同质量浓度白桦脂酸对KYSE170细胞均有抑制作用,抑制效果呈时间和剂量依赖性,24、48、72 h的IC50分别为(5681±256)、(3973±277)和(2928±305)  μg·mL-1;克隆形成结果显示,0、5、20、40 μg·mL-1药物作用细胞后,克隆形成率分别为（8956+500）%、（6100+203）%、（3133+351）%和（1533+233）%,随白桦脂酸质量浓度增大而明显降低（P< 001）。凋亡结果显示, 各实验组KYSE170细胞凋亡率随白桦脂酸质量浓度加大和药物作用时间的延长明显增高（P＜ 005或001）。细胞周期结果显示,0、1、10 μg·mL-1白桦脂酸作用细胞24 h后,随药物质量浓度增大,G1期细胞比例减少,S期细胞比例不断增加,实验组与空白组对比均有统计学差异（P<001）。结论白桦脂酸能抑制人食管鳞癌KYSE170细胞的生长,这一作用可能是通过诱导细胞凋亡、阻滞细胞停留在S期来实现的。     

Cinobufacini,an aqueous extract from Bufo bufo gargarizans Cantor,induces apoptosis through a mitochondria-mediated pathway in human hepatocellular carcinoma cells

Aim of the study

Cinobufacini (Huachansu), an aqueous extract from the skin and parotid venom glands of Bufo bufo gargarizans Cantor, is a traditional Chinese medicine widely used in clinical cancer therapy in China. The present study sought to investigate the possible signaling pathway implicated in cinobufacini-induced apoptosis in the hepatocellular carcinoma cell lines HepG₂ and Bel-7402.

Materials and methods

The effects of cinobufacini on cell proliferation of HepG₂ and Bel-7402 cells were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assays. Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry analysis. The mitochondrial membrane potential (Δψm) and caspase-9 and -3 activity were detected using MitoCapture reagent staining and colorimetric assays, respectively. The expression of apoptosis-related proteins and release of cytochrome c were assessed by Western blot analysis.

Results

Cinobufacini significantly inhibited cell proliferation of both cell lines in a dose- and time-dependent manner. Marked changes in apoptotic morphology and apoptosis rates were clearly observed after cinobufacini treatment. The protein expression of Bax increased whereas that of Bcl-2 decreased, leading to an increase in the Bax/Bcl-2 ratio. Subsequently, cinobufacini disrupted the mitochondrial membrane potential (Δψm) and resulted in the release of cytochrome c, activation of both caspase-9 and -3, and cleavage of poly (ADP-ribose) polymerase (PARP).

Conclusion

The present study indicated that cinobufacini can induce apoptosis of HepG₂ and Bel-7402 cells through a mitochondria-mediated apoptosis pathway.     

Differentiation of bel-7402 human hepatocarcinoma cells induced by aqueous extracts of fresh gecko (AG) and its anti-tumor activity in vivo

Ethnopharmacological relevance

Gecko, a kind of reptile, has been widely used as a traditional Chinese medicine to treat various diseases including cancer in China for thousands of years. The aim of this study was to investigate the anti-tumor effect of AG (aqueous extracts of fresh gecko) on human hepatocellular carcinoma cell Bel-7402 in vitro and mouse H22 hepatocellular in vivo. Further to underlie the molecular mechanism of AG inducing the differentiation of Bel-7402 cells.

Materials and methods

AG was obtained by water extracting method and qualitatively analyzed through High Performance Liquid Chromatography. The total protein concentration of AG was measured by BCA (bicinchoninic acid disodium) assay. The anti-tumor activities in vivo were analyzed through H22 (mouse hepatocellular carcinoma cell line H22) tumor xenografts mice. The cytotoxic activity of AG on Bel-7402 cells was evaluated by MTT assays. AFP (alpha fetoprotein) was detected by radioimmunoassay. ALB (albumin), ALP (alkaline phosphatase) and γ-GT (γ-glutamyl transpeptidase) were detected by biochemical methods with commercial kits. While morphological changes were observed through an inverted microscope. Moreover, the expression level of the proteins involved in MAPK (mitogen-activated protein kinase) signal pathway which was closely related to cellular differentiation was assessed by Western blot.

Results

AG showed obviously anti-tumor activity in vivo and anti-proliferative activity on Bel-7402 cells in vitro both dose-dependently. The number of clones of Bel-7402 cells treated with AG reduced and the cells were displaying differentiation state such as relatively bigger size and dispersed growth. The biochemical function markers of the cells were significantly changed after being treated with AG. The data showed that AFP secretion of the cells decreased 42.5%, ALB secretion increased 58.9%, the activity of ALP and γ-GT markedly decreased 67.0% and 48.5% separately when the concentration of AG was 10 μl/ml, and those effects were all in a dose-dependent manner. The major original and phosphorylated signal proteins (ERK1/2 (extracellular sigal-regualted kinase 1/2), P38 (p38 MAPK) and JNK1/2 (c-Jun N-terminal kinase 1/2)) involved in MAPK signal pathway were measured and the results showed that AG activated the ERK1/2 of Bel-7402 cells.

Conclusions

AG has anti-tumor activity in vivo and inhibits Bel-7402 cell proliferation in vitro through inducing cell differentiation, and the mechanism involves the activation of ERK1/2.     

冬凌草甲素对人肝癌BEL-7402细胞的增殖抑制作用及其作用机制

目的:探讨冬凌草甲素对肝癌BEL-7402细胞的增殖抑制作用及其作用机制。方法:以不同浓度的冬凌草甲素作用于体外培养的BEL-7402细胞,MTT法检测细胞生长抑制率,应用流式细胞仪检测细胞凋亡,Hoechst33258荧光染色法观察细胞凋亡时的形态学变化。应用PCR-ELISA及RT-PCR法检测细胞凋亡前后端粒酶活性及端粒酶hTERTmRNA表达水平的变化。结果:冬凌草甲素可显著的抑制BEL-7402细胞的生长,诱导细胞发生凋亡,并呈现出明显的量-效与时-效关系。冬凌草甲素作用48~60h后在Hoechst33258荧光染色图片上可见核浓缩及核碎裂等典型的细胞凋亡特征,同时端粒酶hTERTmRNA的表达水平及端粒酶活性均明显降低。结论:冬凌草甲素能抑制BEL-7402细胞的生长及诱导细胞发生凋亡;降低端粒酶hTERTmRNA的表达水平及端粒酶活性可能是其重要作用机制之一。     

白花蛇舌草多糖对人宫颈癌Hela细胞和人肝癌Bel-7402细胞生长的影响

目的研究白花蛇舌草多糖对体外培养的人肝癌Bel-7402细胞和人宫颈癌Hela细胞生长的影响。方法常规法体外培养人肝癌Bel-7402细胞和人宫颈癌Hela细胞,设空白对照组、阳性对照组和供试药物组(大、中、小剂量),分别采用MTT法和流式细胞仪检测白花蛇舌草多糖对人宫颈癌Hela细胞和人肝癌Bel-7402细胞的生长抑制率和凋亡率;采用倒置显微镜观察细胞形态学变化。结果白花蛇舌草多糖可抑制人肝癌Bel-7402细胞和人宫颈癌Hela细胞的生长,并促进其凋亡,其作用效果与其浓度正相关,与空白对照组相比具均有显著性差异(P﹤0.01)。结论本研究结果提示,白花蛇舌草多糖具有较好的抗肿瘤活性。