三氧化二砷诱导人肝癌SMMC-7721细胞凋亡及对OPN表达的影响

目的探讨三氧化二砷（As2O3）治疗肝癌的可行性及机制。方法将一定浓度梯度的As2O3与人肝癌细胞株SMMC-7721孵育后,采用MTT法、荧光显微镜及流式细胞仪检测细胞增殖与凋亡变化,逆转录聚合酶链反应（RT-PCR）检测肝癌细胞株中骨桥蛋白基因（OPN mRNA）表达水平。结果As2O3作用后SMMC-7721细胞生长明显受抑,且呈时间-浓度依赖性;荧光显微镜下细胞呈典型的凋亡形态学改变;在流式细胞仪上可见“凋亡峰”,细胞周期阻滞于G2／M期;细胞OPN mRNA表达阳性,OPN mRNA表达水平明显下调。结论As2O3体外能有效抑制肝癌细胞株生长;其机制可能为诱导细胞凋亡、下调OPN mRNA表达。     

JNK/SAPK信号转导系统在三氧化二砷诱导肝癌细胞凋亡中的作用

目的:探讨JNK/SAPK信号转导系统在三氧化二砷(As2O3)诱导人肝癌细胞株HepG2凋亡过程中的作用.方法:采用MTT法观察不同浓度的As2O3对人类肝癌细胞株HepG2细胞生长的抑制作用;以流式细胞术观察细胞的凋亡率及生长周期的变化;以Western blot法检测p-MEK4、JNK、P-JNK、Caspase-3及PARP蛋白在As2O3作用下及SP600125阻断JNK信号转导通路情况下的表达.结果:各浓度As2O3均能明显抑制肝癌细胞HepG,增殖,且具有剂量依赖性和时间依赖性;流式细胞术(FCM)分析显示,As2O3能够诱导肝癌细胞HepG2凋亡且具有时间依赖性,细胞滞留于G2/M期(0,24,48,72 h百分率分别为7.22%±1.50%,11.56%±0.73%,33.8%±1.62%,46.02%±0.11%):Western blotting结果显示,As2O3诱导肝癌细胞HepG2凋亡伴随着Caspase-3和PARP的活化;As2O3作用于HepG2细胞10 min后P-MEK4和P-JNK蛋白表达开始增加,20 min达到高峰,30 min开始减少,总JNK蛋白的含量无明显改变,MEK4和JNK的激活早于细胞凋亡;用SP600125预处理HepG2细胞株后,可以明显减少Caspase-3和PARP的活化.结论:As2O3可以体外通过诱导细胞凋亡抑制肝癌细胞株HepG2的增殖,细胞凋亡通过Caspase-3途径实现.JNK信号转导通路参与了As2O3诱导的HepG2凋亡反应,并位于Caspase-3的上游.     

亚砷酸诱导肝癌细胞株HepG-2细胞凋亡的实验研究

目的探讨亚砷酸(As2O3)对肝癌HepG-2细胞的增殖抑制和诱导凋亡的作用.方法应用细胞计数、FITC-TUNEL染色后荧光摄象及流式细胞仪技术探讨亚砷酸对肝癌HepG-2细胞的增殖抑制及诱导凋亡作用.结果亚砷酸能明显抑制肝癌HepG-2细胞的生长,5μmol/LAs2O3组抑制程度明显大干1μmol/L As2O3组(P＜0.01),二者与不加药阴性对照组相比均有显著性差异(P＜0.01),亚砷酸对肝癌细胞的生长抑制具有时间依赖性,5μmol/L As2O3组已表现出细胞毒作用;1μmol/L As2O3组作用d 3开始出现凋亡细胞,FITC-TUNEL染色后荧光摄象可见典型的凋亡细胞.流式细胞仪可观察到凋亡细胞具有时间依赖性.结论亚砷酸能够抑制肝癌HepG-2细胞的增殖,且能诱导肝癌细胞的凋亡,具有治疗肝癌的潜在价值.     

奥曲肽对人肝癌细胞生长增殖、甲胎蛋白合成及细胞凋亡的作用

目的　研究奥曲肽对肝癌细胞生长增殖及细胞凋亡的作用 ,探讨其对肝癌的作用机制 ,为临床应用提供实验依据。方法　采用MTT法、生长曲线观察奥曲肽对肝癌细胞HepG2生长增殖的影响 ,电化学发光法测定培养上清液中甲胎蛋白 (AFP)含量 ,并用荧光染色、透射电镜和流式细胞仪检测细胞凋亡。结果　奥曲肽在 0 .0 0 5～ 80 μg/ml浓度范围内呈剂量依赖性方式抑制肝癌细胞HepG2的生长增殖 ,并能显著减少肝癌细胞AFP合成。奥曲肽 (0 .2 5～ 4 .0 μg/ml)作用 4 8h后 ,荧光染色与透射电镜可见部分HepG2细胞呈典型的凋亡形态学改变。流式细胞仪检测 ,出现凋亡峰 ,与对照组相比 ,细胞的凋亡率显著升高 (P <0 .0 5 )。结论　奥曲肽能够显著抑制肝癌细胞生长 ,并诱导肝癌细胞凋亡 ,有望成为治疗肝细胞癌的一个有效药物     

三氧化二砷对肝癌细胞株凋亡的诱导作用

目的研究三氧化二砷(As2O2)对人、鼠肝癌细胞株凋亡的诱导作用.方法用不同质量浓度(0.25,0.5,1,2,3,4 mg/L)的三氧化二砷处理体外培养的鼠7919,人HePG2肝癌细胞株,以MTT比色法、AO/EB荧光染色法、DNA凝胶电泳和免疫组化染色法检测细胞凋亡.结果不同浓度的三氧化二砷均可抑制7919,HePG2细胞生长;AO/EB荧光染色在荧光显微镜下观察肝癌细胞呈典型的凋亡形态学改变;DNA凝胶电泳呈梯形条带为凋亡的特征;免疫组化染色加药组bcl-2表达下调,bax表达上调,对照组bcl-2表达上调,bax表达下调,两株细胞结果相同.结论As2O3对两株细胞均有诱导凋亡作用.这为临床应用As2O3治疗肝癌提供了一个可供参考的实验依据.     

三氧化二砷诱导多药耐药肝癌细胞BEL-7402/ADM凋亡的实验研究

目的研究As2O3诱导多药耐药肝癌细胞BEL-7402/ADM凋亡的效果,为As2O3在治疗已产生多药耐药性的原发性肝癌方面的应用提供理论依据。方法采用流式细胞仪检测、分析As2O3诱导BEL-7402/ADM、BEL-7402细胞凋亡情况。结果在体外As2O3能够诱导BEL-7402/ADM、BEL-7402凋亡,其强度与作用浓度和时间有关,两细胞系凋亡率差异无统计学意义。结论As2O3能够诱导表达mdr-1的多耐药细胞BEL-7402/ADM凋亡,将其应用于临床治疗已产生耐药性的原发性肝癌有可能产生较为理想的治疗效果。     

三氧化二砷对肝癌Mahlavu细胞内线粒体跨膜电位的影响

[目的]探讨三氧化二砷（As2O3）作用于肝癌细胞后对其增殖抑制及线粒体跨膜电位（△Ψm）的影响。[方法]用As2O3作用于体外培养人肝癌Mahlavu细胞，用MTT法检测As2O3对人肝癌Mahlavu细胞增殖的影响，并用流式细胞仪观察As2O3对人肝癌Mahlavu细胞△Ψm的影响。[结果]MTT检测显示As2O3能明显抑制人肝癌Mahlavu细胞的增殖，并呈时间和剂量依赖性。流式细胞仪分析显示As2O3作用后的人肝癌Mahlavu细胞内△Ψm明显降低（P〈0．01），并呈时间和剂量依赖性。[结论]As2O3可降低人肝癌Mahlavu细胞△Ψm并可由此诱导细胞凋亡而抑制其增殖。这可能也是As2O3诱导肝癌Mahlavu细胞凋亡的机制之一。     

DHA对人肝癌HepG2细胞的生长抑制作用观察

体外加入二十二碳六烯酸（DHA）培养人肝癌细胞HepG2,MTT法观察细胞生长增殖情况,油红染色、荧光染色、透射电镜观察细胞形态及结构,流式细胞仪分析细胞凋亡率。结果显示,DHA能抑制HepG2生长,呈浓度和时间依赖性（P〈0．01）;细胞内有大量脂滴,并呈典型的凋亡改变,HepG2发生早期凋亡。认为DHA可抑制人肝癌细胞HepG2的生长,并使细胞发生早期凋亡。     

三氧化二砷诱导胃癌细胞凋亡及其对C-myc和TGF-β1表达的影响

目的 探讨三氧化二砷（As2O3）对体外培养的人胃癌细胞SGC-7901生长的抑制和诱导细胞凋亡的作用。方法 用MTT法检测药物效应，透射电镜及流式细胞仪观察As2O3处理SGC-7901细胞后细胞周期细胞凋亡。细胞免疫化学观察As2O3处理后SGC-7901细胞TGF-β1及C-myc表达的变化。结果 ①MTT法证实As2O3对SGC-7901细胞生长有抑制作用，并呈剂量．效应关系。②流式细胞仪分析As2O3处理SGC-7901细胞后细胞滞留于G2／M期，并可见凋亡峰。③透射电镜可见SGC-7901细胞出现典型的细胞凋亡及坏死形态学改变。④As2O3导致C—myc表达的波动，下调TGF-β1，表达。结论As203对人胃癌细胞SGC-7901有抑制作用，其抑制作用可能通过导致C—myc基因表达波动，诱导胃癌细胞凋亡和坏死、阻抑细胞周期进程，抑制SGC-7901细胞增殖。同时通过下调TGF-β1表达阻止胃癌的恶性进程。     

索拉非尼联合三氧化二砷治疗肝癌的研究

目的探讨索拉非尼联合三氧化二砷(As2O3)对肝癌的治疗作用。方法采用MTT实验检测索拉非尼联合As2O3对肝癌细胞的毒性。流式细胞仪检测索拉非尼联合As2O3诱导肿瘤细胞凋亡。构建肝癌异位瘤模型,检测索拉非尼联合As2O3对肝癌荷瘤裸鼠生存期的影响。结果与生理盐水组和单独给药组相比,联合干预组对肝癌具有更强的细胞毒性,差异具有统计学意义(P0.05)。细胞凋亡实验结果表明,联合干预能够促进肿瘤干细胞凋亡,差异具有统计学意义(P0.01)。体内治疗实验结果显示,索拉非尼与As2O3联合干预能够显著延长荷瘤小鼠的中位生存期,诱导肿瘤细胞的凋亡。结论索拉非尼联合As2O3能够增强对肝癌HepG2细胞的增殖抑制作用,是一种潜在的肝癌有效治疗方法。     

氧化砷诱发胃癌细胞株凋亡的初步研究

目的在三氧化二砷（Ａｓ２Ｏ３）治疗ＡＰＬ的基础上，进一步探讨Ａｓ２Ｏ３能否诱发胃癌细胞株凋亡。方法采用荧光标记法，经流式细胞仪和荧光显微镜观察Ａｓ２Ｏ３对ＭＫＮ４５和ＳＧＣ７９０１胃癌细胞株的凋亡诱发率和形态学改变。结果发现Ａｓ２Ｏ３诱发胃癌细胞凋亡率高于５－Ｆｕ的作用；形态学见凋亡细胞呈荧光标记阳性，细胞内出现斑块状荧光，体积缩小。结论Ａｓ２Ｏ３可诱发胃癌细胞凋亡，有必要进一步探索其对胃癌治疗的价值。     

Induction of apoptosis by arsenic trioxide and hydroxy camptothecin in gastriccancer cells in vitro

AIM To study the effects of arsenic trioxide and HCPT on different degrees of differentiated gastric cancer cells (SGC-7901, MKN-45, MKN-28)with respect to both cytotoxicity and induction of apoptosis in vitro. ~ODS The cytotoxicity of As2O3 and HCPT on gastric cancer cells was determined by MTTassay. Morphologic changes of apoptosis of gastric cancer cells were observed by light microscopy and transmission electron microscopy. Apoptosis and cell cycle changes of gastric cancer cells induced by HCPT and As2O3 were investigated by TUNEL method and flow cytometry. RESULTS As2O3 and HCPT had remarkable cytotoxic effects on different degrees of differentiated gastric cancer cells. The IC50 of As2O3 on well differentiated gastric cancer cell MKN-28, moderately differentiated gastric cancer cell SGC-7901, and poorly differentiated gastric cancer cell MKN-28 were 8. 91 μmol/L, 10. 57 μmol/L, and 11.65 μmol/L, respectively. The IC50 of HCPT on MKN-28, SGC-7901, and MKN-45 were 9. 35 rg/L, 10. 21 rg/L, and 12. 63 mg/L respectively after 48 h treatment. After 12 h of exposure to both drugs, gastric cancer cells exhibited morphologic features of apoptosis, including cell shrinkage, nuclear condensation,and formation of apoptotic bodies. A typical subdiploid peak before G0/G1 phase was observed by flow cytometry. The apoptotic rates of SGC7901, MKN-45, and MKN-28 were 13. 84%, 22.52%, and 9. 68%, respectively after 48 h exposure to 10 μmol/L As2O3. The apoptotic rates of SGC-7901, MKN-45, and MKN-28 were 21.88%, 12.35%, and 30. 26%, respectively after 48 h exposure to 10 mg/L HCPT. The apoptotic indice were 7% - 15% as assessed by TUNEL method. The effect of As2O3 on SGC-7901 showed remarkable cell cycle specificity, which induced cell death in G1 phase, and blocked G2/M phase. HCPT also showed a remarkable cell cycle specificity, by inducing cell death and apoptosis in G1 phase and arrest of proliferation at S phase. CONCLUSION AS2O3 and HCPT exhibit significant cytotoxicity on gastric cancer cells by induction of apoptosis. As2O3 and HCPT might have a promising prospect in the treatment of gastric cancer, which needs to be further studied.     

氧化砷诱导胃癌细胞凋亡的实验研究

目的 研究三氧二化砷（氧化砷）对胃癌细胞的诱导凋亡作用。方法 应用ＴＵＮＥＬ染色、流式细胞仪技术研究氧化砷对胃癌细胞ＭＫＮ－４５、ＭＫＮ－２８的诱导凋亡作用。结果 氧化砷作用于不同分化程度的胃癌细胞后,可看到较为典型的细胞凋亡的形态学变化：细胞核固缩,染色质凝集,呈新月型紧贴于核膜周边,核碎裂,染色质片断化,凋 亡小体形成等。流式细胞仪ＤＮＡ直方图上出现典型的亚二倍体的“凋亡峰”。ＴＵＮＥＬ染色法     

药物诱导胃癌细胞凋亡的初步探索

目的:了解羟基喜树碱和氧化砷体外诱导胃癌细胞凋亡的能力.探索最佳诱导时间和剂量.并初步阐明其作用机制。方法:利用HE染色法、流式细胞仪和DNA末端原位标记染色法(TUNEL)观察羟基喜树碱和氧化砷在体外对胃癌细胞MKN-28(高分化腺癌)。SGC-7901(中分化腺癌)。MKN-45(低分化腺癌)的作用 结果:药物作用48小时后,羟基喜树碱0.01mg/ml组胃癌细胞MKN-28、SGC-7901、MKN-45的凋亡率分别为30.26％、21.88％和12.35％,氧化砷10μmol/L组胃癌细胞MKN-28、SGC-7901、MKN-45的凋亡率分别为22.52％、13.83％和9.68％其中羟基喜树碱在细胞周期的S期诱导胃癌细胞发生凋亡,而氧化砷则主要作用于G2/M期。     

Synergistic effect of cell differential agent-II and arsenic trioxide on induction of cell cycle arrest and apoptosis in hepatoma cells

AIM: To illustrate the possible role of cell differential agent-II (CDA-II) in the apoptosis of hepatoma cells induced by arsenic trioxide (As(2)O(3)). METHODS: Hepatoma cell lines BEL-7402 and HepG2 were treated with As(2)O(3) together with CDA-II. Cell surviving fraction was determined by MTT assay; morphological changes were observed by immunofluorescence staining of Hoechst 33,258; and cell cycle and the apoptosis index were determined by flow cytometry (FCM). RESULTS: Cytotoxicity of CDA-II was low. Nevertheless, CDA-II could strongly potentiate arsenic trioxide-induced apoptosis. At 1.0 g/L CDA-II, IC(50) of As(2)O(3) in hepatoma cell lines was reduced from 5.0 micromol/L to 1.0 micromol/L (P<0.01). The potentiation of apoptosis was dependent on the dosage of CDA-II. FCM indicated that in hepatoma, cell growth was inhibited by CDA-II at lower concentrations (<2.0 g/L) primarily by arresting at S and G(2) phase, and at higher concentrations (>2.0 g/L) apoptotic cell and cell cycle arresting at G(1) phase increased proportionally. The combination of two drugs led to much higher apoptotic rates, as compared with the either drug used alone. CONCLUSION: CDA-II can strongly potentiate As(2)O(3)-induced apoptosis in hepatoma cells, and two drugs can produce a significant synergic effect.     

Synergistic effect of cell differential agent-Ⅱ and arsenic trioxide on induction of cell cycle arrest and apoptosis in hepatoma cells

AIM: To illustrate the possible role of cell differential agent-Ⅱ (CDA-Ⅱ) in the apoptosis of hepatoma cells induced byarsenic trioxide (As2O3).METHODS: Hepatoma cell lines BEL-7402 and HepG2 weretreated with As2O3 together with CDA-Ⅱ. Cell survivingfraction was determined by MTT assay; morphologicalchanges were observed by immunofluorescence staining ofHoechst 33 258; and cell cycle and the apoptosis index weredetermined by flow cytometry (FCM).RESULTS: Cytotoxity of CDA-Ⅱ was low. Nevertheless, CDA-Ⅱ could strongly potentiate arsenic trioxide-inducedapoptosis. At 1.0 g/L CDA-Ⅱ, IC50 of As2O3 in hepatoma celllines was reduced from 5.0 μmol/L to 1.0 μmol/L (P＜0.01).The potentiation of apoptosis was dependent on the dosageof CDA-Ⅱ. FCM indicated that in hepatoma, cell growth wasinhibited by CDA-Ⅱ at lower concentrations (＜2.0 g/L)primarily by arresting at S and G2 phase, and at higherconcentrations (＞2.0 g/L) apoptotic cell and cell cyclearresting at G1 phaseincreased proportionally. Thecombination of two drugs led to much higher apoptotic rates,as compared with the either drug used alone.CONCLUSION: CDA-Ⅱ can strongly potentiate As2O3-induced apoptosis in hepatoma cells, and two drugs canproduce a significant synergic effect.     

氧化砷抗胰腺癌作用及其机制的实验研究

目的:探讨氧化砷(As_2O_3)体外体内抗胰腺癌作用及其可能机制。方法:应用MTT比色法、流式细胞仪、裸鼠肾包膜下移植瘤抑制试验和细胞形态学方法进行检测。结果:As_2O_3抑制胰腺癌细胞生长并具有时间与浓度依赖性,6天组抑制50％细胞生长的药物浓度(IC_(50))为0.625～1.25μg/ml。癌细胞在As_2O_3作用下,G0/G1期细胞比例、凋亡指数(AI)增高,增殖指数(PI)下降,AI/PI比例增大。As_2O_3能显著抑制肾包膜下胰腺癌移植瘤生长。电镜检查证实As_2O_3诱导的胰腺癌细胞具有凋亡与分化特征。结论:As_2O_3抗胰腺癌细胞作用主要通过诱导细胞凋亡与分化而得到体现。     

槲皮素对胃癌细胞SGC-7901和BGG-823生长的影响

目的 观察槲皮素对胃癌细胞SGC-7901和BGC-823生长和增殖的影响。方法 以台盼蓝拒染法计数胃癌细胞的生长抑制率，荧光显微镜了解凋亡的发生，流式细胞术检测细胞周期变化。结果 台盼蓝拒染法计数显示槲皮素抑制SGC-7901和BGC-823细胞增殖的作用明显，呈浓度和时问依赖性，槲皮素处理48h后的Ic50为14．12μm(SGC-7901)和28．13μm(BGC-823)。形态学检测显示出细胞凋亡的特征变化，流式细胞仪检测表明经10～20μm／L的槲皮素处理，SGC-7901细胞周期阻滞于G0／G1期，BGC-823细胞周期阻滞于S期。结论 槲皮素能抑制胃癌细胞的生长并诱导其发生凋亡，是有效的抗癌药。     

Effect of arsenic trioxide on rat hepatocarcinoma and its renal cytotoxicity

AIM: To study the effect of arsenic trioxide (As2O3) on rat experimental hepatocarcinoma and its renal cytotoxicity.METHODS: The hepatocarcinoma model was established by diethaylnitrosamine perfusion in stomach of 120 Wistar rats, and the treatment began at the end of 20 weeks.Before the treatment, the rat models were randomly divided into 5 groups. In the treatment groups, three doses of As2O3 were injected into rat abdominal cavity, the total time of drug administration was 4 weeks. Cisplatin control or the blank group was injected into abdominal cavity with equal amount of cisplatin or saline at the same time,respectively. On the 7th, 14th and 28th day after the treatment, the hepatocarcinoma nodules were obtained and the morphologic changes of hepatocarcinoma cells were observed under light and electron microscopes;Immunohistochemistry (S-P methods) was employed to detect the expression of bcl-2, bax and PCNA in hepatocarcinoma tissues; flow cytometry (TUNEL assay)was used to detect the apoptosis of liver cancer cells and the change of cytokinetics. On the 28th day, the kidneys were obtained and their histologic changes were observed under light microscope, and immunohistochemistry (SP stain) was also employed to detect the expression of bcl-2and PCNA. Cisplatin and saline solution were used as the control.RESULTS: As2O3 could induce the apoptosis of rat liver cancer cells and exhibited typical morphologic changes.The incidence of apoptosis of hapatocarcinoma cells was elevated (P=0.001). The elevation was the most higher in the group of middle-dose of As2O3 (1 mg.kg-1), significantly higher than that of the other arsenic groups and the controls (P=0.001). Large dose of As2O3 (5 mg.kg-1) was able to arise the incidence of apoptosis, but also produced a large amount of necrosis and inflammatory reaction. Middle dose of As2O3 dramatically increased the cell number in G2/M phase (P=0.0001), and apoptosis happened apparently.The expression of bcl-2 and bax was related to the dose of As2O3. With the up-regulation of apoptotic incidence, the ratio of bcl-2/bak decreased. But the incidence of apoptosis was not the highest status and the ratio of bcl-2/bax was at the lowest when the highest-dose of As2O3 was used.There was significant difference among the PCNA indexes (PCNA L1) of the five groups. Of them, three arsenic groups all showed decrease of different degrees, and this downregulation was most obvious in group A. There was significant difference among the three groups (P=0.016).Under the light microscope, the rat kidney in the cisplatin group exhibited tubular epithelium swelling and degeneration, protein casts in collecting tubules; While all arsenic groups didn't show the significant changes (P=0.013).In the arsenic groups, the expression of bcl-2 in the renal tubular epithelium was increased (P=0.005), no obvious changes happened to PCNA L1. But in the group of cisplatin,the PCNA L1 increased significantly (P=0.001).CONCLUSION: AS2O3 can induce apoptosis of rat hepatocellular carcinoma cells. And there is optimum dose;too high dose will induce the cytotoxic effect, while certain dose of As2O3 is able to block the cell cycle at G2/M phase.As2O3 had the most remarkable influence on G2/M cells,and it can also induce apoptosis to cells at other phases.As2O3 can restrain the proliferation of rat hepatocellular carcinoma cells, in a dose-time dependent manner.Compared with cisplatin, As2O3 didn't show obvious renal toxicity, which was related to the increasing expression of bcl-2 in renal tubular epithelium, the inhibition of apoptosis and the anti-oxidation effects.