酒石酸锑钾在诱导人肝癌BEL-7402细胞凋亡中的影响

目的:本研究旨在明确酒石酸锑钾(PAT)在体外对人肝癌BEL7402细胞凋亡的影响及抑癌机制。方法:用PAT以不同浓度、不同时间作用于人肝癌BEL7402细胞,以诱导其凋亡。用MTT比色法观察其细胞毒性,荧光显微镜、透射电镜、TUNEL染色法及流式细胞术(FCM)等方法来检测凋亡,观察其形态学和生化方面的变化。结果:PAT以剂量依赖和时间依赖的方式抑制BEL7402细胞的生长。5～40μmol·L-1的PAT处理48h后,形态学上,肝癌细胞表现为细胞皱缩、核质浓缩、核碎裂、细胞起泡以及凋亡小体形式等凋亡特征的形态学改变。DNA末端原位标记染色法、流式细胞仪均能检测到凋亡细胞。结论:PAT在体外诱导肝癌BEL7402细胞凋亡,能作为一种凋亡诱导剂用于肝癌的治疗。     

Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II A.

In order to study the effect of tanshinone II A on growth and apoptosis in human hepatoma cell line BEL-7402 in vitro, the human hepatoma cell line BEL-7402 was treated with tanshinone II A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC50 value being 6.28 micrograms/ml. After treatment with 1-10 micrograms/ml tanshinone II A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 micrograms/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32 +/- 0.16)%, (3.01 +/- 0.35)%, (3.87 +/- 0.43)%, (6.73 +/- 0.58)% and (20.85 +/- 1.74)% respectively, which were all significantly higher than those in the control group (1.07 +/- 0.13)%. It is concluded that Tanshinone II A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition.     

亚砷酸体外对人肝癌细胞株BEL-7402影响的初步研究

目的 体外培养人肝癌细胞株BEL－7402,从多个角度探讨三氧化二砷（As2O3）的抗肿瘤作用及其机制。方法 应用倒置相差显微镜、电子显微镜、透谢电镜、流式细胞仪,分别对不同浓度加药组及对照组BEL－7402细胞的存活。形态学改变,细胞DNA含量的分布进行了观察和测定。结果 0.5、1、2μmol/L As2O3均能抑制人肝癌细胞株BEL－7402细胞的生长增殖。流式细胞仪分析显示,加药组在G1期细胞前均出现亚二倍体峰,且G0／G1期细胞减少,S期细胞增多;电镜下,对照组细胞核质比大、核大、核膜有明显切迹,0.5μmol/L As2O3组细胞核质比减少、核变圆、胞浆内出现分化良好的细胞器, 0.5、1、2μmol/L As2O3组均可见细胞膜完整、核固缩、凋亡小体形成。结论 三氧化二砷不仅抑制人肝癌细胞增殖,而且诱导细胞凋亡。     

Effect of tanshinone IIA on the growth behavior of human hepatoma cell line BEL-7402 in vitro and its mechanism]

OBJECTIVE: To investigate the effect of tanshinone IIA on the growth behavior of human hepatoma cell line BEL-7402 in vitro and explore the mechanism. METHODS: Human hepatoma cell line BEL-7402 was exposed to tanshinoneIIA at different concentrations for 72 h, and the suppression of the cell growth was observed under inverted-phase contrast microscope. Apoptosis-related alterations in the cell morphology and biochemistry were examined under fluorescence microscope and transmission electron microscope (TEM) and by DNA agarose gel electrophoresis, and the apoptotic rate was quantified by flow cytometry (FCM). RESULTS: After treatment with 0-10 microg/ml tanshinone IIA for 72 h, the proliferation of BEL-7402 cells was significantly suppressed, and cell apoptosis occurred characterized by cell shrinkage, nuclear chromatin condensation and fragmentation, formation of membrane blebs and apoptotic bodies as observed under fluorescence microscope and TEM. DNA ladder was presented in DNA electrophoresis. FCM analysis yielded the cell apoptotic rates of (20.78+/-2.17) %, (24.64+/-2.07) %, (31.47+/-3.86) %, (43.65+/-4.04) % and (52.36+/-3.75) % at tanshinone IIA concentrations of 0.5, 1.0, 2.0, 5.0 and 10.0 microg/ml respectively, all significantly higher than those of the control group [(2.37+/-0.29)%]. CONCLUSION: Tanshinone IIA can inhibit the growth of human hepatoma BEL-7402 cells possibly through the mechanism of apoptosis induction.     

Growth inhibition and apoptosis induction in human hepatoma cells by tanshinone II<Subscript>A</Subscript>

Summary In order to study the effect of tanshinone II_A on growth and apoptosis in human hepatoma cell line BEL-7402in vitro, the human hepatoma cell line BEL-7402 was treated with tanshione I_A at various concentrations for 72 h. Growth suppression was evaluated by MTT assay; apoptosis-related alterations in morphology and biochemistry were ascertained under cytochemical staining (Hoechst 33258), transmission electron microscopy (TEM), and DNA agarose gel electrophoresis. Apoptotic rate was quantified by flow cytometry (FCM). The results showed that Tanshinone II_A could inhibit the growth of hepatoma cells in a dose-dependent manner, with IC₅₀ value being 6.28 μg/ml. After treatment with 1–10 μg/ml tanshione II_A for 72 h, BEL-7402 cells apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies were observed. DNA ladder could be demonstrated on DNA electrophoresis. FCM analysis showed hypodiploid peaks on histogram, and the apoptotic rates at 5 μg/ml concentration for 12 h, 24 h, 36 h, 48 h and 72 h were (2.32±0.16)%, (3.01±0.35)%, (3.87±0.43)%, (6.73±0.58)% and (20.85±1.74)% respectively, which were all significantly higher than those in the control group (1.07±0.13)%. It is concluded that Tanshione II_A could induce human hepatoma cell line BEL-7402 apoptosis, which may be related to the mechanism of growth inhibition. TANG Zhongzhi, male, born in 1966, Doctor in Charge This project was supported by a grant from Natural Sciences Foundation of Hubei Province (No. 2000J064).     

Antineoplastic mechanism of Octreotide actionin human hepatoma

Objectives To investigate whether apoptosis can be induced by Octreotide in human hepatoma cells in vitro and elucidate the antineoplastic mechanism of Octreotide in hepat oma. Methods A cultured human hepatoma cell line, BEL-7402, was exposed to Octreotide and ap optosis was evaluated by cytochemical staining (Hochesst 33 258), transmiss ion electron microscopy, agarose gel electrophoresis and flow cytometry (FCM).Results After exposure to 0.2 μg/ml Octreotide, apoptosis with nuclear chromatin cond ensation as well as fragmentation, cell shrinkage and the formation of apoptotic bodies was observed using cytochemical staining and transmission electron micros copy. A DNA ladder in agarose gel electrophoresis was also displayed. FCM show ed that the apoptotic cell number rose with an increase in the concentration of Octreotide (0-2 μg/ml). There was a positive correlation between Octreotide concentration and apoptotic rate in BEL-7402 cells (r=0.809, P&lt;0.05) .Conclusion Apoptosis in human hepatoma cells can be induced by Octreotide, which may be rel ated to the mechanism of antineoplastic action ofOctreotide in hepatoma.     

反义IGF-Ⅰ寡核苷酸转染对人肝癌细胞增生及凋亡的影响

为研究胰岛素样生长因子 I(IGF I)反义寡核苷酸转染对人肝癌细胞增生、分化及凋亡的影响 ,探讨寡核苷酸转染治疗肝癌的可行性 ,利用反义核酸技术 ,合成针对IGF I的寡核苷酸片段 ,利用脂质体包裹反义IGF I寡核苷酸片段瞬时转染人肝癌细胞系BEL 74 0 2细胞 ,MTT法检测细胞增生 ;放免法检测培养细胞上清中AFP、CEA的分泌量 ;采用末端标记 (Tunel)法检测细胞凋亡的变化。结果发现转染反义IGF I寡核苷酸可使人肝癌细胞系BEL 74 0 2细胞增生下降 ,AFP、CEA表达降低 ,凋亡细胞数量增多。反义IGF I寡核苷酸转染人肝癌细胞系BEL 74 0 2细胞可以降低细胞增生、减少细胞去分化并诱导肝癌细胞凋亡     

Growth Inhibition and Apoptosis Induction in Human Hepatoma Cells by Tanshinone Ⅱ_A

Summary:In order to study the effect of tanshinone Ⅱ_A on growth and apoptosis in human hepatomacell line BEL-7402 in vitro,the human hepatoma cell line BEL-7402 was treated with tanshinone Ⅱ_Aat various concentrations for 72 h.Growth suppression was evaluated by MTT assay;apoptosis-relat-ed alterations in morphology and biochemistry were ascertained under cytochemical staining(Hoechst33258),transmission electron microscopy(TEM),and DNA agarose gel electrophoresis.Apoptoticrate was quantified by flow cytometry(FCM).The results showed thst Tanshinone Ⅱ_A could inhibitthe growth of hepatoma cells in a dose-dependent manner,with IC_(50) value being 6.28μg/ml.Aftertreatment with 1—10 μg/ml tanshinone Ⅱ_A for 72 h,BEL-7402 cells apoptosis with nuclear chro-matin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodieswere observed.DNA ladder could be demonstrated on DNA electrophoresis.FCM analysis showedhypodiploid peaks on histogram,and the apoptotic rates at 5     

Effect of lidamycin on telomerase activity in human hepatoma BEL-7402 cells

Objective To investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence. Methods Chromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-β-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA. Results Mitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-β-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells. Conclusion Mitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.     

反义人端粒酶RNA诱导肝癌细胞凋亡的分子生物学机制研究

目的 探讨反义人端粒酶RNA( human telomerase RNA,hTR)基因对肝癌细胞株Bel-7402凋亡的诱导作用及其分子生物学机制.方法 利用前期实验成功转染人端粒酶正、反义RNA基因的肝癌细胞,分为正义转染组(Bel-7402-hTR-EcoRI)、反义转染组(Bel-7402-hTR-BamHI)和空白对照组(Bel-7402).PCR鉴定转染结果,hoechst 33258荧光染色观察3组细胞的变化,实时荧光定量PCR法和Western blot法分别检测凋亡相关基因p53、Bcl-2和Bax的mRNA和蛋白的表达水平.结果 与正义转染组及空白对照组细胞相比,反义转染组部分细胞出现凋亡特征性改变,p53、Bax的mRNA和蛋白表达水平明显升高(P＜0.05),Bcl-2的mRNA和蛋白表达水平明显降低(P＜0.05).结论 反义端粒酶RNA可诱导肝癌细胞凋亡,其机制可能是通过上调p53和Bax的表达并下调Bcl-2的表达.     

紫杉醇、5-氟脲嘧啶抑制肝癌细胞BEL-7402生长和诱导凋亡的比较

OBJECTIVE: To investigate the effect of paclitaxel and 5-flurouracil (5-Fu) on growth inhibition and apoptosis of human hepatoma BEL-7402 cells. METHODS: Growth inhibition of BEL-7402 cells treated with paclitaxel and 5-Fu, respectively, was measured by ATP-tumor chemosensitivity assay (ATP-TCA), and the cell cycle kinetics and apoptosis were analyzed by flow cytometry and microscopic examination. RESULTS: BEL-7402 cells were highly sensitive to paclitaxel with growth inhibition observed in both dose- and time-dependent manners (IC(50)=5.58 x 10(-7) mol/L). Paclitaxel induced significantly higher rate of cell apoptosis than the control group (P<0.05) but significantly lower rate than that induced by 5-Fu (P<0.01). Necrosis was observed predominantly in paclitaxel-treated cells whereas 5-Fu caused mainly cell apoptosis (P<0.05). Levels of apoptosis increased in proportion to the decrement of paclitaxel concentration but directly proportional to increment of 5-Fu concentration. CONCLUSIONS: Paclitaxel and 5-Fu are effective in inducing growth inhibition and apoptosis of BEL-7402 cells. While 5-Fu causes mainly apoptosis in hepatoma cells, the anticancer mechanism of paclitaxel is predominantly through induction of necrosis.     

紫杉醇、5-氟脲嘧啶抑制肝癌细胞BEL-7402生长和诱导凋亡的比较

目的 研究紫杉醇、5-氟脲嘧啶(5-Fu)抑制肝癌细胞BEL-7402增殖和诱导凋亡的效果。方法 采用ATP生物发光法检测肝癌细胞株的药物敏感性,以流式细胞术、形态学方法分析细胞凋亡和细胞周期。结果 BEL-7402对紫杉醇高度敏感,对5-Fu低度敏感,抑制作用均存在剂量、时间效应。紫杉醇组3种浓度诱导细胞凋亡率均明显高于无药对照组(P<0.05),但低于5-Fu组(P<0.01);紫杉醇组细胞死亡率显著高于5-Fu组(P<0.05)。紫杉醇诱导凋亡率随药物浓度降低而增加,而5-Fu诱导凋亡率随药物浓度降低而降低。结论 紫杉醇可抑制BEL-7402肝癌细胞增殖和诱导凋亡。5-Fu抑癌以诱导细胞凋亡为主,紫杉醇则以引起细胞坏死为主。     

根皮素诱导肝癌BEL-7402细胞凋亡

目的 研究根皮素诱导肝癌细胞系BEL-7402细胞凋亡.方法 MTT法测定BEL-7402细胞毒性,荧光显微镜观察细胞形态学的变化,流式细胞仪分析细胞周期和线粒体膜电位的变化.发色底物法检测Caspase-3、Caspase-6和Caspase-9活性变化.结果 根皮素对BEL-7402细胞IC50在89.23 μg/mL.BEL-7402细胞生长曲线表明,根皮素浓度增高,生长率明显下降.细胞凋亡可在40～160 μg/mL根皮素处理后24 h出现.凋亡细胞主要表现为核染色质固缩,荧光染色增强.根皮素阻断细胞于G1期,线粒体膜电位降低.Caspase-3、Caspase-6和Caspase-9被激活,呈时间依赖性改变.根皮素处理BEL-7402细胞12 h Caspase-9活性最高,而Caspase-6活性在18 h达峰值,Caspase-3活性峰值时间在24h后.结论 根皮素可以诱导BEL-7402细胞发生凋亡,途径可能是通过线粒体旁路.     

小檗碱对人肝癌Bel-7402细胞增殖和凋亡的影响

目的观察小檗碱对人肝癌Bel-7402细胞增殖抑制及诱导凋亡的作用.方法体外培养人肝癌Bel-7402细胞,台盼兰活细胞计数及集落形成抑制实验观察不同浓度小檗碱对Bel-7402细胞的增殖抑制作用,Hochest33258染色观察凋亡形态学变化,流式细胞仪分析细胞周期及凋亡率.结果小檗碱可显著抑制Bel-7402细胞生长,使集落形成能力明显下降,均呈时间、剂量依赖性.小檗碱处理后72 h,Hochest33258染色可观察到细胞核边集、固缩,有明显凋亡小体形成,流式细胞仪检测出现明显Sub-G1峰.结论小檗碱可以抑制人肝癌Bel-7402细胞增殖,诱导细胞凋亡,小檗碱可能具有治疗人肝细胞癌的潜在应用价值.     

三氮唑席夫碱衍生物对肝癌细胞裂亡的影响

目的:研究3-吡啶-3-基-4-[(4-甲氧基-苯亚甲基)氨基]-5-甲硫基-1,2,4-三唑(LH-38)对肝癌细胞BEL-7402裂亡的作用。方法:BEL-7402细胞常规培养于RPMI-1640培养液中,细胞生长至对数生长期加LH-38(终浓度分别为1×10-4mol/L和1×10-5mol/L),连续培养48 h或72 h。四甲基偶氮唑蓝(MTT)比色法检测细胞增殖,荧光染料Hoechst33258和PI联染检测细胞死亡,免疫细胞化学法检测激活型Caspase-3表达。结果:LH-38抑制BEL-7402细胞增殖并呈浓度依赖关系,IC50为3.0×10-4mol/L;1×10-5mol/L浓度的LH-38处理细胞72 h,镜下可见多倍体细胞明显增多,可见微核或多核细胞染色体自发性的凝集,有丝分裂异常,存活或死亡的多核或单核巨细胞同时存在,存活细胞的激活型Caspase-3表达阴性;1×10-4mol/L浓度LH-38处理细胞48 h,可明显诱导细胞凋亡。结论:不同浓度LH-38可以引起人肝癌细胞BEL-7402细胞裂亡或细胞凋亡,即两种不同形式的细胞死亡。     

RNAi沉默STAT3基因联合mTOR抑制剂rapamycin诱导BEL-7402肝癌细胞凋亡

目的：探讨PI3K/AKT/mTOR和JAK/STAT3 2条信号转导途径共同作用对肝癌细胞凋亡的影响,为肝癌基因治疗提供依据。方法：选取对数生长期BEL-7402细胞,随机分为对照组、mTOR抑制剂rapamycin(Rapa)组、阴性质粒组、阴性质粒+ Rapa组、STAT3-siRNA质粒组和STAT3-siRNA 质粒+Rapa组,应用LipofectamineTM 2000转染试剂将含有目的基因的质粒转染BEL-7402细胞,同时应用rapamycin,分别采用流式细胞术和Hoechst33258荧光染色检测细胞凋亡率和形态学的变化,JC-1 荧光染色观察线粒体膜电位(ΔΨm)变化,Western blotting法检测活性caspase-3蛋白表达水平。结果：STAT3-siRNA+Rapa组细胞凋亡率为60.22%±0.87%,明显高于其他各组(P<0.05),且细胞ΔΨm明显降低(27.28%±1.82%,P<0.05);Hoechst33258荧光染色检测,见STAT3-siRNA有大量细胞出现细胞核聚集、边缘化和核
碎裂等典型细胞凋亡形态;Western blotting检测,STAT3-siRNA+Rapa组活性caspase-3蛋白表达水平明显高于其他各组（P<0.05）。结论：RNAi沉默BEL-7402肝癌细胞STAT3基因联合rapamycin可促进BEL-7402肝癌细胞的凋亡,二者具有明显的协同作用。     

绿脓杆菌制剂对人肝癌细胞株BEL-7402的杀伤效应

目的探索绿脓杆菌制剂对人肝癌细胞BEL-7402的杀伤效应。方法采用MTT法检测不同浓度的绿脓杆菌制剂对人肝癌细胞BEL-7402增殖的作用,同时利用电子显微镜、透射电镜观察细胞BEL-7402的形态学变化。结果MTT检测表明：绿脓杆菌制剂为10×107/mL、5×107/mL、2.5×107/mL时对肝癌细胞生长杀伤作用与对照组比较差异有高度统计学意义（P〈0.01）;电子显微镜、透射电镜观察发现肝癌细胞BEL-7402于12、24 h出现凋亡形态学改变,40 h形态学表现为凋亡与坏死并存。结论绿脓杆菌制剂对肝癌细胞BEL-7402生长有抑制作用,诱导细胞凋亡及坏死可能是主要作用机制。     

HL-60细胞凋亡小体体外负载树突状细胞的实验研究

目的 :证实树突状细胞 (Dendritic cells,DCs)可通过吞噬凋亡小体获取抗原物质 ,探讨其在肿瘤免疫治疗中的意义。方法 :利用吸附单克隆抗体 -磁珠分离系统 (MACS) ,从人脐血中分离出 CD34+ 细胞 ,体外以重组 h GM- CSF+ h SCF+ h TNF-α+ h FL诱导培养 DCs,光镜观察诱生细胞的形态 ,流式细胞术 (FACS)检测其表型。电镜观察和流式细胞仪检测三氧化二砷 (As2 O3 )诱导凋亡的 HL - 6 0细胞 ,将凋亡的 HL- 6 0细胞与培养早期的 DCs共育 4～ 8h,电镜观察 DCs吞噬凋亡小体的现象。结果 :CD34+细胞经诱生后生成了大量具有典型形态和表型的成熟 DCs;电镜下见凋亡的 HL- 6 0细胞内凋亡小体形成 ,流式细胞仪检测 DNA含量 ,见亚二倍体峰形成。凋亡细胞与 DCs共育后 ,电镜下见 DCs吞噬凋亡小体的现象。结论 :DCs可通过吞噬凋亡小体摄取抗原物质。     

急性胰腺炎大鼠胰腺细胞凋亡分析方法的比较

目的探讨牛磺胆酸钠所诱导的急性胰腺炎急性期胰腺细胞凋亡现象的特点和作用。方法大鼠造模、分组、取材后采用电镜定性检测，DNA末端原位染色法检测（TUNEL）和流式细胞仪定量检测（FCM）。结果①电镜检测：对照组胰腺腺泡细胞超微结构正常；水肿组自噬空泡形成，凋亡细胞和凋亡小体出现。坏死组早期凋亡和坏死可以并存，且凋亡现象较水肿组明显：②TUNEL检测：除了在3h时段坏死与对照组间差异非常显著（P〈0．01）、水肿与对照组间差异显著（P〈0．05）外，其余组间差异均无显著性，但以坏死组凋亡现象最为明显：③FCM检测：TUNEL与FCM检测结果呈正相关。结论凋亡只是一种代偿，可能减轻病情的严重程度，但他只是在一定限度内发挥着积极作用。     

体外联合培养条件下小鼠睾丸足细胞诱导成熟T淋巴细胞凋亡

目的 :探讨体外联合培养条件下睾丸足细胞能否诱导 T淋巴细胞凋亡。方法 :用 TU NEL 标记、核酸电泳、电镜、流式细胞术检测对照组 (T淋巴细胞 )、A组 (T淋巴细胞 +培养睾丸足细胞 2 4h培养基上清 )、B组 (T淋巴细胞 +睾丸足细胞 )等 3组中小鼠成熟 T淋巴细胞的凋亡及其数量的变化。结果 :3组 T淋巴细胞在电镜下均可见细胞染色质浓集 ,核固缩裂解 ,凋亡小体形成 ;TUNEL 标记见部分细胞核染色呈深蓝色 ;核酸电泳出现典型 DNA梯状带 ;流式细胞术检测 A,B两组 T淋巴细胞凋亡量均明显高于对照组 (P<0 .0 1) ,B组 T淋巴细胞凋亡数量显著高于 A组 (P<0 .0 1)。 结论 :体外培养条件下小鼠睾丸足细胞能够诱导成熟 T淋巴细胞凋亡。