全反式维A酸、阿维A和他扎罗汀对人黑色素瘤细胞A375凋亡和Bax/Bcl-2蛋白表达的影响及意义

目的 探讨全反式维A酸(ATRA)、阿维A及他扎罗汀对人黑色素瘤细胞A375凋亡及Bax/Bcl-2蛋白表达的影响及其意义。方法 采用体外培养和流式细胞仪检测细胞凋亡(AnnexinV-PI法),SABC免疫细胞化学方法检测细胞Bax/Bcl-2蛋白表达情况。结果 浓度均为10^-5mol/L的ATRA、阿维A及他扎罗汀能不同程度地诱导了人黑色素瘤细胞A375凋亡,其中阿维A作用最为显著,凋亡率13.42%,明显高于对照组、ATRA及他扎罗汀组(均P<0.05);其次为ATRA(5.03%,明显高于对照组及他扎罗汀组,P<0.05)和他扎罗汀组(凋亡率2.88%)。3种维A酸亦使A375细胞Bax蛋白阳性表达增强而Bcl-2蛋白阳性表达减弱(P<0.05),其中阿维A作用最强,其次为ATRA和他扎罗汀。结论 维A酸促进A375细胞凋亡可能部分通过以线粒体为核心的凋亡途径。阿维A可能是防治黑色素瘤的有效药物。     

放射诱导鼠脑神经元凋亡

目的 探讨X射线诱导鼠脑神经元凋亡与照射剂量的关系及时间规律。方法 将大鼠分为不同剂量照射组和对照组,以双标法计数凋亡的神经元数。结果 X射线能诱导的鼠脑神经元的凋亡,凋亡率在照射前后有显著性差异(P<0.0001),在各剂量组间亦有显著性差异(P<0.005),同剂量组在照射后不同时间点间亦有显著性差异。结论 低、中剂量X射线可诱导鼠脑神经元凋亡,凋亡率有剂量依赖性并具有时间规律性。     

星形胶质细胞条件培养液对tbOOH致PC12细胞凋亡的抑制作用

目的 探讨星形胶质细胞条件培养液对活性氧叔丁基脂氢过氧化物tbOOH诱导的PC12细胞凋亡的影响。方法 以分离、纯化的SD大鼠大脑皮层星形胶质细胞制备的条件培养液培养tbOOH应激的PC12细胞,采用荧光显微镜和透射电镜形态观察细胞凋亡;用流式细胞术测定细胞凋亡率的变化;用巴比妥酸法评估细胞内丙二醛含量的变化。结果 tbOOH可诱导PC12细胞凋亡,而星形胶质细胞条件培养液可降低其凋亡;同时,巴比妥酸法显示星形胶质细胞条件培养液可显著性降低PC12细胞丙二醛含量(P<0.05)。结论 星形胶质细胞条件培养液有提高PC12细胞抗氧化能力,可抑制活性氧tbOOH诱导的神经细胞凋亡。     

肝刺激因子稳定转染人肝癌细胞系的方法建立与表达鉴定

目的 建立稳定表达肝刺激因子(hepatic stimulator substance,HSS)的BEL-7402肝癌细胞系,并对其生物学特性进行鉴定。方法 采用脂质体法将鉴定后的Flag-HSS-pcDNA3.0质粒转染到肝癌细胞系BEL-7402中,进行G418筛选,获得稳定转染的人肝癌细胞系。通过real-time PCR、 Western blotting和免疫荧光染色等方法鉴定HSS mRNA和蛋白质表达及亚细胞定位。结果 免疫荧光染色实验证实HSS基因能在稳定转染的肝癌细胞系中表达,并定位于线粒体中。同时发现在稳定转染细胞中HSS mRNA和蛋白质的表达也均高于野生型BEL-7402细胞。结论 目前已成功构建稳定转染HSS的BEL-7402肝癌细胞系。为深入研究HSS的生理功能提供实验工具和研究基础。     

血流阻断的缺血预处理技术在肝癌切除术中的应用

目的 探讨缺血预处理(ischemic preconditioning, IP)在血流阻断的伴有肝硬化的肝癌切除中的保护作用机制及临床应用价值。方法 将本院手术切除的34例原发性肝癌患者随机分为2组,IP组18例,肝门阻断切肝前先给予缺血5 min、灌注5 min的缺血预处理;对照组16例,单纯肝门阻断切肝,2组手术由同一组医师完成。比较2组患者手术前后肝功能的变化和肝灌注1 h时肝组织Fas-mRNA表达、Caspase-3活性变化和细胞凋亡的情况。结果 术后1、3、7 d,IP组的血清天门冬氨酸氨基转移酶、丙氨酸氨基转移酶水平明显低于对照组(t=6.985, P<0.01);术后3、7 d,IP组的总胆红素明显低于对照组(t=3.447, P<0.05);术后1 d,IP组的白蛋白高于对照组(t=3.360, P<0.05)。术后1 h,IP组肝组织Fas-mRNA表达、Caspase-3活性和细胞凋亡均明显低于对照组(t=3.771, P<0.05)。结论 IP对肝癌患者入肝血流阻断肝切除术后的肝功能有良好的保护作用,其保护机制是通过下调Fas-mRNA表达和抑制Caspase-3的活性,从而抑制肝细胞凋亡,该技术简便易行值得临床推广应用。     

白凤菜总黄酮对多发性骨髓瘤U266细胞增殖和凋亡影响

目的 观察白凤菜总黄酮(TFG)对多发性骨髓瘤U266细胞的增殖和凋亡的影响。方法 MTT实验检测U266细胞增殖;倒置显微镜及荧光显微镜分别观察U266细胞经TFG处理24 h后细胞形态及凋亡的变化;流式细胞术检测细胞凋亡及细胞周期的变化。结果 TFG明显抑制U266细胞的增殖,P<0.05,且呈浓度和时间依赖性;100 μg /mLTFG作用组的U266细胞凋亡明显增加;TFG使U266细胞周期阻滞于S期。结论 TFG可抑制U266细胞增殖,可能同细胞周期的捕获和细胞凋亡的诱导相关。     

紫杉醇和吉西他滨对激素非依赖性前列腺癌的作用

目的 观察紫杉醇(PA)协同吉西他滨(GE)对前列腺癌细胞系PC-3的体内外作用,并探讨其可能的作用机制。方法 应用光镜形态学、噻唑蓝(MTT)法、流式细胞仪和免疫细胞化学法观察了10^-6、10^-7、10^-8 mol/L浓度PA和10^-7、10^-8、10-9 mol/L浓度GE在体外单药或协同对前列腺癌细胞系PC-3的作用、对细胞DNA含量及cyclin D1表达的影响。观察PC-3细胞荷瘤裸鼠单独及协同使用PA和GE前后的体质量、肿瘤质量、血清PSA和肿瘤PSA免疫组化的变化。结果 10^-8 mol/L以上浓度GE作用48 h,可增强10^-7 mol/L以上浓度PA对前列腺癌PC-3细胞系的生长抑制[抑制率≥(50.8±4.2)%,P<0.05],增强诱导凋亡作用[凋亡率≥(22.9±2.3)%,P<0.05],下调Cyclin D1的表达[表达率≤(9.6±1.6)%],与阳性对照组cyclin D1表达率(25.5±4.1)%相比差异有显著性(P<0.01)。GE使PA所致的G₂/M期细胞周期阻滞比例由(70.3±9.7)%减至(38.2±4.2)%,部分地逆转了其G₂/M期细胞周期阻滞(P<0.01)。协同治疗前后裸鼠体质量无明显变化,但肿瘤质量(3.2.±0.5 g)、血清PSA[(51±14) ng/ml]和肿瘤PSA免疫组化的表达率[(30±3.7)%]在协同治疗组显著低于其他组(P<0.05或0.01)。结论 PA和GE可以在体内外协同增强对前列腺癌细胞系PC-3的生长抑制和诱导凋亡作用,显示了PA和GE协同用于治疗激素非依赖性前列腺癌的可能性,并部分地解释了其作用机制。     

生物活性玻璃45S5体外对根尖牙乳头细胞成牙本质方向分化的影响

目的 明确生物活性玻璃(45S5)对人根尖牙乳头细胞体外成牙本质方向分化的作用。方法 浓度为0.1 mg/mL的45S5浸提培养液与人根尖牙乳头细胞共培养,离子体发射光谱检测45S5浸提培养液硅、钙、磷离子浓度,细胞活性噻唑蓝比色法检测细胞增殖,Realtime-PCR检测细胞成牙本质方向分化相关基因牙本质涎磷蛋白(DSPP)、牙本质基质蛋白(DMP-1)的表达,茜素红矿化结节染色及氯化十六烷基吡啶钙结节半定量分析矿化结节形成。结果 与对照组相比,45S5浸提培养液中硅离子浓度显著升高(P<0.05);45S5浸提培养液连续诱导培养根尖牙乳头细胞3、5、7 d后,与对照组相比显著促进细胞增殖(P<0.05);45S5浸提培养液连续诱导培养根尖牙乳头细胞7 d,细胞DSPP、DMP-1的表达量显著高于对照组(P<0.05);45S5生物活性玻璃体浸提培养液连续诱导培养根尖牙乳头细胞14和21 d,茜素红染色后观察45S5组和矿化诱导培养液(OM)组均见明显红染矿化结节,对照组未见明显红染矿化结节;氯化十六烷基吡啶钙结节半定量分析各组钙离子浓,45S5和OM组OD值高于对照组(P<0.05),45S5组OD值高于OM组(P<0.05)。结论 0.1 mg/mL生物活性玻璃45S5体外促进根尖牙乳头细胞增殖、成牙本质方向分化相关基因高表达和矿化结节形成,45S5体外促进根尖牙乳头细胞成牙本质方向分化。     

光气对小鼠肺上皮细胞和内皮细胞凋亡的诱导作用

目的 研究光气对肺水肿小鼠肺上皮细胞和内皮细胞凋亡的诱导作用。方法 将32只BALB/C小鼠随机分成正常对照组(16只)和染毒组(16只)2组。正常对照组小鼠以空气为对照,染毒组小鼠给予11.9mg/L剂量的光气,时间为5min。染毒后4h,取小鼠分离、培养肺Ⅱ型细胞用于电镜观察和流式细胞仪检测细胞凋亡,取小鼠肺脏进行DNA琼脂糖凝胶电泳和TUNEL染色检测肺组织凋亡。结果 光气染毒肺水肿小鼠电镜下原代肺Ⅱ型细胞出现凋亡小体;流式细胞术显示肺Ⅱ型细胞凋亡率(40.26±7.74)显著高于正常对照组(1.58±1.01,P<0.001);DNA琼脂糖凝胶电泳出现"DNA梯状改变";TUNEL染色检测到肺上皮细胞和血管内皮细胞凋亡。结论 光气染毒可造成肺水肿小鼠肺脏细胞发生凋亡,提示光气造成小鼠肺水肿的发生机制存在细胞凋亡的诱导作用。     

黄芩苷体外抑制氟尿嘧啶耐药肝癌细胞的生长及作用机制

目的:观察黄芩苷体外对氟尿嘧啶(Fu)耐药肝癌细胞株生长的影响,并探讨其可能的作用机制.方法:体外培养人肝癌细胞BEL-7402及其耐药细胞BEL-7402/5-Fu,MTT法观察黄芩苷作用后细胞增殖情况;流式细胞术测定细胞内罗丹明123荧光强度;RT-PCR检测细胞多重耐药MDR1基因表达;Western印迹法检测细胞P-gP蛋白表达;应用Matrigel模型测定细胞黏附率;荧光免疫技术测定细胞β1-整合素(β1-integrin)和上皮钙黏附素(E-CD)蛋白表达.结果:黄芩苷明显抑制肝癌细胞BEL-7402及其耐药细胞BEL-7402/5-Fu的增殖,IC50分别为34.2、36.6 mg/L.5、10 mg/L黄芩苷能部分逆转耐药细胞对Fu的耐药,逆转倍数分别为28.6、46.7;5、10 mg/L黄芩苷增强BEL-7402对Fu敏感性,增敏倍数分别为1.4、2.1.5、10 mg/L黄芩苷增加耐药细胞内药物积聚,降低MDR1基因及P-gP蛋白表达,抑制耐药细胞黏附率,降低耐药细胞β1-整合素表达,促进E-CD蛋白表达;且10 mg/L黄芩苷效果优于5 mg/L(P均＜0.05).结论:黄芩苷体外能抑制肝癌细胞生长及降低细胞黏附性,并能部分恢复肝癌耐药细胞对Fu的敏感性,这可能与其增加细胞内药物浓度、抑制MDR1基因表达有关.     

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.     

绿芦笋对体外人肝癌细胞生长的影响

在体外培养人肝癌细胞（ＢＥＬ－７４０２）过程中，加入自制的绿芦笋原汁和绿芦笋乙醇提取液，同时选用５－氟脲嘧啶和三尖杉酯碱作为阳性药物，观察对细胞生长的抑制作用。每天每组取３瓶细胞进行活细胞计数，连续１０ｄ并绘制生长曲线，结果表明，低浓度的绿芦笋原汁和绿芦笋乙醇提取液（２０ｍｇ／ｍｌ）能显著抑制肝癌细胞的生长。     

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.     

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。     

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。     

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。     

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     

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.     

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).     

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.