1,25-二羟基维生素D3和维生素K2联合诱导HL-6O细胞分化及凋亡的研究

目的研究1,25-二羟基维生素D3[1,25(OH)2D3,简称D3]与维生素K2(VK2)联合应用对HL-60细胞分化及凋亡的影响.方法通过四唑氮蓝(MTT)比色,细胞形态,流式细胞仪(FCM)测定细胞周期、凋亡率及CD14的表达,观察D3、VK2对HL-60细胞的影响.结果D3与VK2都能抑制HL-60细胞增殖,并且联合使用抑制作用显著.10-8mol/L D3与10 μmol/L VK2联合处理HL-60细胞72 h后CD14的表达率为63.15%,且G0/G1期细胞显著增多,与单独一种比较差异有统计学意义(P＜0.05).10 μmol/L、20 μmol/L VK2作用于HL-60细胞72 h凋亡率分别为11.31%、20.36%,与对照组比较差异有统计学意义(P＜0.01);而10 μmol/LVK2与10-8 mol/L D3联用72 h细胞的凋亡率为5.41%,与对照组比较差异无统计学意义(P＞0.05).结论D3与VK2联合使用可以使D3诱导分化作用加强,而VK2诱导凋亡作用受到抑制.     

益气活血软坚解毒含药血清诱导人肝癌细胞系Bel-7402细胞的凋亡

目的:研究益气活血软坚解毒(YHRJ)含药血清对人肝癌细胞系Bel-7402生长抑制及诱导凋亡作用.方法:将YHRJ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:YHRJ含药血清具有抑制Bel-7402肿瘤细胞生长作用(其中20%YHRJ等效剂量抑制率49.1%,与NS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.流式细胞术检测结果,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:YHRJ含药血清有抑制人肝癌细胞系Bel-7402细胞生长并有诱导细胞凋亡作用.     

氧化砷对胃癌细胞的细胞毒和诱导凋亡作用

目的:研究三氧二化砷(氧化砷)对胃癌细胞的细胞毒和诱导凋亡作用。方法:应用MTT、TUNEL染色、流式细胞仪技术研究氧化砷对不同分化程度的胃癌细胞的细胞毒和诱导凋亡的作用。结果:氧化砷对不同分化程度的胃癌细胞均具有较强的细胞毒作用.10μmol/L氧化砷作用24小时细胞杀伤率为24.6％ 48小时接近50％,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,染色质疑集.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”TU-NEL染色法显示,细胞凋亡指数为1.4％～9.5％。氧化砷的细胞毒作用呈时间和剂量依赖性,且表现为细胞周期特异性,作用48小时后,SGC-7901细胞的细胞周期变化明显,G0/G1期细胞从54.2％下降到17.7％ 而G2/M期细胞则从20.2％上升到63.4％,说明氧化砷主要作用于细胞周期的G2/M期,抑制细胞增殖和诱导细胞凋亡。结论:氧化砷对胃癌细胞具有较强的细胞毒和诱导细胞凋亡的作用.具有治疗胃癌的潜在价值。值得进一步研究     

斑蝥酸钠维生素B6对人喉癌Hep-2细胞增殖的抑制作用

目的探讨斑蝥酸钠维生素B6对人喉癌细胞株(Hep-2细胞)增殖抑制率、凋亡率及其细胞周期进程的影响。方法应用四甲基偶氮唑蓝(MTT)比色法检测斑蝥酸钠维生素B6对Hep-2细胞增殖抑制率、流式细胞仪定量检测经斑蝥酸钠维生素B6作用后的Hep-2细胞周期进程变化及凋亡率。结果不同浓度的斑蝥酸钠维生素B6注射液对Hep-2细胞增殖均有抑制作用,且呈剂量依赖性。流式细胞仪结果显示,G1期细胞增多,S期细胞减少,G2/M期细胞相对增多,Hep-2细胞凋亡率升高。结论斑蝥酸钠维生素B6对人喉癌Hep-2细胞生长、增殖有明显的抑制作用,抑制Hep-2细胞G1期向S期转化进程,从而使S期细胞减少,G2/M期细胞相对增多,诱导Hep-2细胞凋亡。     

芒果甙对肝癌细胞增殖的抑制和凋亡的诱导

目的 观察芒果甙对人肝癌细胞株BEL-7404的毒性和诱导凋亡作用及对细胞周期的阴滞作用，探讨芒果甙作为肿瘤化学预防药物的可能性，方法 用MTT法观察芒果甙对人肝癌细胞株增殖的抑制作用。用光学显微镜观察芒果甙对细胞的毒性作用。以流式细胞仪检测芒果甙对细胞凋亡的诱导作用和对细胞周期的干预作用。结果 不同浓度的芒果甙在不同时间对肝癌细胞株均有毒性作用，并随浓度升高和作用时间的延长而毒性作用增强，凋亡也随之增加，芒果甙阻滞肝癌细胞周期于G2/M期，20μmol/L芒果甙作用24h后上述效果开始明显。结论 芒果甙对肝癌细胞株有明显的毒性作用。能诱导肝癌细胞凋亡和阻滞细胞周期于G2/M期具有潜在药用价值。值得进一步深入研究。     

三氧化二砷诱导肝癌HepG2细胞凋亡作用

目的 探讨三氧化二砷（As2O3）诱导肝癌HepG2细胞凋亡作用。方法 应用普通光学显微镜、荧光显向镜和流式细胞仪观察As2O3对HepG2细胞株的形态学改变和诱发凋亡率。结果 As2O3作用于细胞后,可看到较典型的细胞凋亡形态学改变,细胞体积缩小,染色质固综合成斑块状,呈半月型紧贴于核膜周边,核碎裂,凋亡小体形成等,AO/EB荧光染色法显示。细胞凋亡率为4.3%-9.1%.As2O3诱导HepG2细胞凋亡作用呈时间依赖性,最佳浓度为2umol/L。流式细胞仪DNA直方图上呈现典型的亚二倍体凋亡峰。As2O3主要作用于细胞周期的G2/M期。结论 As2O3具有诱导HepG2细胞凋亡作用,存在治疗肝癌的潜在价值。     

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。     

三氧化二砷对慢性髓系白血病细胞周期及Survivin表达的影响

目的研究三氧化二砷(As2O3)对慢性髓系白血病细胞(K562)的作用特点及其机制。方法不同浓度As2O3作用于K562细胞后，四唑盐(MTT)比色法分析细胞增殖；流式细胞术检测细胞周期分布、细胞凋亡及Survivin抗原表达；RT-PCR检测Survivin mRNA的表达。结果2—10μmoL／L的As2O3能有效抑制K562细胞增殖，但未能诱导明显的细胞凋亡。周期分析显示，G2／M期细胞比例显著增多；同时G2／M细胞周期依赖性表达的Survivin mRNA和蛋白表达增加。结论As2O3明显抑制K562细胞的生长，其机制是诱导G2／M期细胞周期停滞。Survivin表达上调可能是K562细胞对As2O3诱导凋亡抵抗的机制之一。     

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.     

三氧化二砷对Hep-2细胞体外生物学行为的影响及相关机制

目的探讨三氧化二砷(As2O3)制剂对人喉癌细胞株(Hep-2)生长、增殖、凋亡及细胞周期进程等生物学的影响。方法四甲基偶氮唑蓝(MTT)比色法检测不同浓度的As2O3对Hep-2细胞的增殖抑制率,流式细胞仪检测细胞周期进程的变化,透射电子显微镜检测Hep-2亚细胞水平改变。结果不同浓度的As2O3对Hep-2细胞增殖抑制率分别为18.8%、31.9%和43.5%,与对照组相比差异显著。流式细胞仪结果显示G0/G1期细胞增多,S期细胞减少,G2/M期细胞相对增多,电子显微镜下可见,线粒体肿胀,大量空泡形成,核染色质趋边、凝集,可见凋亡小体改变;结论As2O3能显著抑制Hep-2细胞的增殖,抑制Hep-2细胞G1期向S期转化进程,诱导Hep-2细胞凋亡及亚细胞结构改变。     

Anti-proliferation and radiosensitization effects of chitooligosaccharides on human lung cancer line Hep G2

Objective: To observe the anti-proliferation and radiosensitization effect of chitooligosaccharides(COS) on human lung cancer cell line Hep G2. Methods: CCK-8 assay was employed to obtain the inhibition ratio of COS on Hep G2 cells at 24 h after treatment. The clonogenic assay was used to analyze the cell viability of RAY group and RAY+COS group with X-ray of 0, 1, 2, 4, 6 and 8 Gy, and the cell survival curve was used to analyze the sensitization ratio of COS. Flow cytometry was employed to detect cell cycle and apoptosis rate in control group, RAY group and RAY+COS group after 24 h treatment. Results: COS inhibited the proliferation of Hep G2 cells, and the inhibition rate positively correlated with the concentration of COS. The cell viability decreased with increasing exposure dose in RAY group and RAY+COS group. The cell viabilities of RAY+COS group were lower than those of RAY group at the dose of 4, 6 and 8 Gy(P0.05), and the sensitization ratio of COS was 1.19. There were higher percentage at G2/M phase and apoptosis rate, and lower percentage at S phase in RAY+COS group versus the other two groups(P0.01). Conclusions: COS can inhibit the proliferation of Hep G2 cells, and enhance the radiosensitization of Hep G2 cells, induce apoptosis and G2/M phase arrest.     

蝙蝠葛活性成分对人肺癌A549细胞株抗增殖作用的实验研究

目的探讨蝙蝠葛活性成分对人肺癌A549细胞株抗增殖作用及其机制。方法应用MTT法测定蝙蝠葛活性成分对人肺癌A549细胞株的生长抑制作用;通过吖碇橙（AO）/溴化乙啶（EB）染色荧光显微镜观察肿瘤细胞的形态学变化;采用流式细胞仪检测A549细胞的周期分布相;应用免疫细胞化学技术SP法检测药物处理前后增殖细胞核抗原Ki-67、Bcl-2的表达。结果蝙蝠葛活性成分对人肺癌A549细胞株有明显的抑制生长的作用,且呈现出浓度依赖性;蝙蝠葛活性成分可诱导A549细胞发生细胞周期阻滞;蝙蝠葛活性成分作用后Ki-67和Bcl-2阳性表达率较对照组降低（P〈0.01）。结论蝙蝠葛活性成分在体外对人肺癌A549细胞株有显著的抑制增殖作用,可能与下调Ki-67、Bcl-2蛋白表达,细胞周期发生G0/G1期阻滞有关。     

Scutellaria barbate extract induces apoptosis of hepatoma H22 cells via the mitochondrial pathway involving caspase-3

AIM： To study the growth inhibitory and apoptotic effects of Scutellaria barbata D.Don （S. barbata） and to determine the underlying mechanism of its antiturnor activity in mouse liver cancer cell line H22.METHODS： Proliferation of H22 cells was examined by MTT assay. Cellular morphology of PC-2 cells was observed under fluorescence microscope and transmission electron microscope （EM）. Mitochondrial transmembrane potential was determined under laser scanning confocal microscope （LSCM） with rhodamine 123 staining. Flow cytometry was performed to analyze the cell cycle of H22 cells with propidium iodide staining. Protein level of cytochrome C and caspase-3 was measured by semi-quantitive RT-PCR and Western blot analysis. Activity of caspase-3 enzyme was measured by spectrofluorometrv.RESULTS： M-I-I- assay showed that extracts from S. barbata （ESB） could inhibit the proliferation of H22 cells in a time-dependent manner. Among the various phasesof cell cycle, the percentage of cells in S phase was significantly decreased, while the percentage of cells in G1 phase was increased. Flow cytometry assay also showed that ESB had a positive effect on apoptosis. Typical apoptotic morphologies such as condensation and fragmentation of nuclei and blebbing membrane of apoptotic cells could be observed under transmission electron microscope and fluorescence microscope. To further investige the molecular mechanism behind ESB-induced apoptosis, ESB-treated cells rapidly lost their mitochondrial transmembrane potential, released mitochondrial cytochrome C into cytosol, and induced caspase-3 activity in a dose-dependent manner. CONCLUSION： ESB can effectively inhibit the proliferation and induce apoptosis of H22 cells involving loss of mitochondrial transmembrane potential, release of cytochrome C, and activation of caspase-3.     

Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line

Abstract: Melatonin reduces proliferation in many different cancer cell lines. However, studies on the oncostatic effects of melatonin in the treatment of hepatocarcinoma are limited. In this study, we examined the effect of melatonin administration on HepG2 human hepatocarcinoma cells, analyzing cell cycle arrest, apoptosis and mitogen‐activated protein kinase (MAPK) signalling pathways. Melatonin was dissolved in the cell culture media in 0.2% dimethyl sulfoxide and administered at different concentrations for 2, 4, 6, 8 and 10 days. Melatonin at concentrations 1000–10,000 μm caused a dose‐ and time‐dependent reduction in cell number. Furthermore, melatonin treatment induced apoptosis with increased caspase‐3 activity and poly(ADP‐ribose) polymerase proteolysis. Proapoptotic effects of melatonin were related to cytosolic cytochrome c release, upregulation of Bax and induction of caspase‐9 activity. Melatonin treatment also resulted in increased caspase‐8 activity, although no significant change was observed in Fas‐L expression. In addition, JNK 1,‐2 and ‐3 and p38, members of the MAPK family, were upregulated by melatonin treatment. Growth inhibition by melatonin altered the percentage or cells in G0–G1 and G2/M phases indicating cell cycle arrest in the G2/M phase. The reduced cell proliferation and alterations of cell cycle were coincident with a significant increase in the expression of p53 and p21 proteins. These novel findings show that melatonin, by inducing cell death and cell cycle arrest, might be useful as adjuvant in hepatocarcinoma therapy.     

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.     

亚砷酸对肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响

目的探讨亚砷酸（AA）对人肝癌BEL-7402细胞增殖、凋亡及其Bcl-2表达的影响。方法采用MTT比色法检测从作用后的BEL-7402细胞增殖抑制率,流式细胞术检测BEL-7402细胞周期及凋亡细胞,HE染色法观察凋亡细胞的形态,RT-PCR检测BEL．7402细胞的Bcl-2 mRNA,免疫组化法检测细胞的Bcl-2蛋白。结果1．0—8．0μmol／L的AA可使BEL-7402细胞增殖抑制率上升,能诱导BEL-7402细胞凋亡并阻滞细胞周期于S、G2／M期,呈剂量依赖性;8．0μmol／L的AA作用BEL-7402细胞48h后,细胞呈现明显的凋亡形态改变,其Bcl-2 mRNA及蛋白表达明显减弱。结论AA体外有抑制BEL-7402细胞增殖及诱导凋亡的作用,且呈时间、剂量依赖性,其作用机制可能与降低其Bcl-2表达有关。     

原白头翁素衍生物对人乳腺癌MCF-7细胞的抗增殖与诱导凋亡作用

目的探讨原白头翁素衍生物（溴甲基呋喃酮）体外对乳腺癌MCF-7细胞株的抗增殖及诱导凋亡作用。方法应用MTT法、倒置显微镜、HE染色、扫描电镜技术、吖啶橙/溴化乙锭荧光染色方法以及流式细胞技术检测原白头翁素衍生物对体外培养的人乳腺癌MCF-7细胞的诱导凋亡及细胞形态学改变。结果MTT法示不同浓度原白头翁素衍生物对MCF-7细胞株均有抗增殖作用,并呈时间—浓度依赖性。倒置显微镜、HE染色、扫描电镜观察示核固缩、碎裂、凋亡小体形成等典型形态学改变。AO/EB双染色结果显示细胞死亡主要以凋亡为主。流式细胞示随着药物浓度的增加细胞凋亡率亦逐渐升高,S期细胞所占比例增大。结论原白头翁素衍生物在体外对人MCF-7乳腺癌细胞株有抗增殖及诱导凋亡作用。     

维生素A对大鼠实验性肺气肿肺泡壁细胞增殖与凋亡的影响

目的 观察维生素A对大鼠实验性肺气肿的干预作用,探讨其对肺泡壁细胞增殖和凋亡的影响.方法 雄性Wistar大鼠24只,采用随机数字表法分为对照组、模型组和维生素A组,每组8只.模型组、维生素A组在实验之初采用气管内滴入2 kU/kg弹性蛋白酶复制肺气肿模型,对照组气管内滴入等量生理盐水,术后第5周起进行药物干预,维生素A组每天给予100 kU/kg维生素A灌胃,其他两组给予等量油剂,共进行4周.实验第8周后处死大鼠.行HE染色观察各组大鼠肺泡的病理学变化,免疫组织化学法观察增殖细胞核抗原(PCNA)的表达,末端脱氧核酸转移酶介导的dUTP缺口末端标记(TUNEL)法观察肺泡壁细胞的凋亡.统计学处理采用单因素方差分析,多组间两两比较采用LSD法.结果 模型组平均肺泡数(43±11)明显低于对照组(101±15)和维生素A组(56±8);平均肺泡面积[(3763±504)μm2]明显高于对照组[(1919±270)μm2]和维生素A组[(2710±276)μm2];增殖指数[(30.7±7.6)%]明显高于对照组[(9.9±1.8)%],明显低于维生素A组[(45.4±5.0)%];凋亡指数[(22.0±4.6)%]明显高于对照组[(9.8±1.7)%]和维生素A组[(17.3±3.5)%];增殖指数/凋亡指数(1.03±0.19)与对照组(1.45±0.52)无明显差别,明显低于维生素A组(2.73±0.64).结论 维生素A能促进弹性蛋白酶诱导大鼠实验性肺气肿的肺泡壁细胞增殖并抑制其凋亡,使肺泡壁细胞增殖/凋亡的平衡向增殖倾斜,从而对大鼠实验性肺气肿起到一定的改善作用.