去甲斑蝥素诱导人肾癌786-0细胞凋亡的体外研究

目的探讨去甲斑蝥素（NCTD）诱导人肾癌细胞株786—0凋亡的机制。方法实验分NCTD组和对照组．分别应用MTT法、流式细胞术和实时定量PCR检测NCTD对体外培养786—0细胞的生长抑制率、细胞周期和凋亡率以及肿瘤凋亡控制因子Bcl-2、Bax mRNA的表达。结果NCTD能抑制786—0细胞生长,随着NCTD浓度升高、处理时间延长作用增强,有明显的时间-剂量依赖性。流式分析显示,786—0细胞的S期细胞减少,G2/M期细胞增多,凋亡率上升。实时定量PCR结果表明,在80μmol／L NCTD处理24h后,Bcl-2 mRNA表达量明显减少、Bax mRNA表达水平则升高,Bcl-2／Bax比值明显下降,与对照组比较差异有统计学意义（P〈0．05）。结论NCTD可明显抑制786—0细胞的增殖和生长,诱导其凋亡,其机制可能与干扰786—0细胞生长周期、抑制DNA合成代谢以及影响凋亡相关基因Bcl-2和Bax的表达有关。     

肿瘤细胞裂解物抗小鼠B16F10黑色素瘤的作用研究

反复冻融B16F10肿瘤细胞制备裂解物,以白喉毒素(Diphtheria toxin,DT)为载体,OK432和来源于结核分枝杆菌(Mycobacterium tuberculosis)热休克蛋白70(HSP70)第407-426(mHSP70407～426,M)的两段串联重复序列M2为佐剂,制备了肿瘤细胞疫苗B16F10-DT-M2-OK432(BDTMOK),探讨其能否抑制小鼠B16黑色素瘤,并且对其抗肿瘤的作用机理进行部分探讨。以制备的BDTMOK免疫C57BL/6小鼠,分别检测体液免疫应答和细胞免疫应答。通过ELISA法,从血清中检测到高滴度的抗B16肿瘤细胞裂解物(B16 tumor cell lysate,B16TCL)类抗体。淋巴细胞增殖实验的结果显示,BDTMOK的免疫能够有效的刺激脾淋巴细胞的增殖。预防结合治疗性实验的结果显示,BDTMOK激发的免疫应答对于B16肿瘤攻击起到有效的保护作用,与PBS阴性对照组比较,皮下注射BDTMOK可以延长皮下移植瘤发生的潜伏期(P<0.05),并且平均瘤重显著降低(P<0.05);抑制了小鼠皮内肿瘤模型中的血管新生(P<0.01)。疫苗BDTMOK能有效的抑制小鼠B16黑色素瘤的生长。     

珠子参总皂苷对H2O2诱导新生大鼠心肌细胞凋亡的抑制作用

目的:观察珠子参总皂苷对H2O2诱导新生大鼠心肌细胞凋亡的抑制作用,并探讨其作用机制.方法:Wistar乳鼠心肌细胞原代培养,用H2O2建立氧化应激损伤模型,然后用珠子参总皂苷(100,200 μg/mL)孵育24 h后,MTT法检测珠子参总皂苷对细胞活力的影响,流式细胞仪和Hoechst33258染色检测珠子参总皂苷对氧化应激诱导细胞凋亡及胞内ROS含量的影响,比色法测定心肌细胞Caspase-3、Caspase-9的活性,荧光定量PCR测定心肌细胞Bcl-2和Bax mRNA表达,并计算Bcl-2与Bax的比值.结果:珠子参总皂苷(100,200 μg/mL)能显著改善心肌细胞活性,有效保护线粒体膜电位的稳定,抑制心肌细胞凋亡和改善细胞形态,降低细胞内活性氧(ROS)含量;下调Bax mRNA表达,上调Bcl-2mRNA表达及Bcl-2与Bax的比值;降低H2O2所致新生大鼠心肌细胞中Caspase-3、Caspase-9的活性.结论:珠子参总皂苷对H2O2诱导心肌细胞凋亡有显著的抑制作用,其机制可能与其稳定心肌细胞膜、清除ROS及调节心肌细胞Bcl-2、Bax和Caspase-3、Caspase 9表达有关.     

诺美孕酮促进大鼠子宫内膜异位症细胞凋亡及其作用机制

目的探讨诺美孕酮促进实验性大鼠子宫内膜异位症模型细胞凋亡及其作用机制。方法外科手术法建立大鼠子宫内膜异位症模型;HE染色观察异位内膜病理组织学改变;ELISA测定血清中抗子宫内膜抗体(EMAb)水平;West-ern blot检测异位内膜组织中Bcl-2、Bax、Caspase-9、Caspase-3蛋白的表达情况。结果诺美孕酮(0.5、1.5、15 mg.kg-1)异位内膜萎缩,间质疏松,腺体数目明显减少,腺腔萎缩;诺美孕酮呈剂量依赖性地降低异位内膜厚度和EMAb的浓度;诺美孕酮(0.5、1.5、15 mg.kg-1)异位内膜组织中Bax、Caspase-9和Caspase-3蛋白表达升高、Bcl-2蛋白表达降低。结论诺美孕酮(0.5、1.5、15 mg.kg-1)可促进大鼠子宫异位内膜细胞凋亡,其作用机制可能与降低EMAb水平、上调Bax和下调Bcl-2蛋白表达、激活细胞凋亡的内源性途径启动因子Caspase-9蛋白、激活执行因子Caspase-3蛋白表达有关。     

姜黄素诱导食管癌Eca-109细胞凋亡机制的研究

目的观察姜黄素诱导食管癌Eca-109细胞凋亡,并初步探讨其作用机制。方法通过CCK-8法检测不同浓度、不同时间点作用于Eca-109细胞的增殖抑制率。应用Caspase 3活性检测试剂盒检测胱天蛋白酶3活性。流式细胞技术检测细胞早期凋亡。免疫细胞化学法检测凋亡相关基因Bax和Bcl-2的表达情况。结果随浓度增加时间延长各组生长抑制率有明显增高,且呈剂量浓度依赖性,组间差异有显著性(P<0.01)。胱天蛋白酶3活性检测发现:姜黄素20μmol·L-1,40μmol·L-1作用24 h后OD值与对照组比较,差异具有统计学意义(P<0.05)。流式细胞术检测到姜黄素作用后,细胞凋亡率明显升高。免疫细胞化学染色显示,随着姜黄素浓度的增高和作用时间的增长,Bcl-2蛋白表达减弱,Bax蛋白表达增强。结论姜黄素可能是通过诱导胱天蛋白酶3表达活性增高,上调促凋亡基因Bax及下调抗凋亡基因Bcl-2的表达,诱导Eca-109发生凋亡。     

雷公藤内酯醇诱导非小细胞肺癌细胞凋亡及其机制初探

目的:研究雷公藤内酯醇(Triptolide,TPL)对非小细胞肺癌细胞A549细胞增殖抑制作用和促凋亡影响的分子机制。方法:以A549细胞为研究对象,采用MTT法测定细胞增殖;吖啶橙/溴乙啶(AO/EB)荧光染色法检测细胞凋亡;Caspase3活性检测试剂盒分析其凋亡机制。结果:在6.25～100 ng·ml~(-1),12～72 h范围内,TPL对A549细胞增殖具有明显抑制作用(P<0.01),48 h内其凋亡率随着时间延长而增加,Caspase3活性早期较对照组有显著提高(P<0.05),并于12 h达到最高值。结论:TPL能有效抑制A549细胞的增殖,这种抑制作用可能与TPL促进肿瘤细胞凋亡,激活Caspase3活性有关。     

黄癸素诱导小鼠黑色素瘤B16细胞凋亡的研究

目的探讨黄癸素诱导小鼠黑色素瘤B16细胞凋亡的作用。方法应用MTT法检测黄癸素对体外培养的B16细胞增殖的抑制作用,观察量效及时效关系;通过Rhodamine123染色、琼脂糖凝胶电泳、Hoechst33258染色、caspase-3和caspase-8活性检测,观察黄癸素诱导细胞凋亡的作用和机制。结果黄癸素抑制B16细胞的增殖,具有明显的时间依赖性和浓度依赖性,作用24、48、72h的IC50分别为16.59、9.29和6.22mg·mL-1;B16细胞经黄癸素处理后,出现染色质固缩、DNALadder等凋亡表现,Rhodamine123染色荧光降低和caspase-3、caspase-8活性增强,提示黄癸素引起线粒体膜电位降低可能触发caspases级联反应而导致细胞凋亡。结论黄癸素可抑制B16细胞增殖,诱导B16细胞凋亡。     

褪黑素诱导NB4细胞凋亡及机制的研究

目的探讨褪黑素对NB4细胞凋亡的增殖抑制和凋亡诱导作用。方法将0、10~(-8)、10~(-6)、10~(-4)mol/L褪黑素在体外与NB4细胞共同培养,应用噻唑蓝(MTT)比色法测定细胞活性,Hoechst荧光染色检测细胞凋亡,流式细胞技术检测凋亡细胞比例,分光光度计检测上清液中Caspase-3及Caspase-9含量。结果褪黑素能显著抑制NB4细胞增殖,促进细胞凋亡,增加凋亡因子的表达。结论褪黑素促进NB4细胞凋亡的机制可能与增加Caspase-3及Caspase-9表达有关。     

蓝萼甲素对宫颈癌HeLa细胞的作用及其相关机制研究

目的观察蓝萼甲素对宫颈癌HeLa细胞的增殖抑制作用,探讨其诱导凋亡的机制。方法采用体外细胞培养方法,以不同浓度蓝萼甲素作用于HeLa细胞,用四甲基偶氮唑盐法(MTT法)检测细胞的存活率;透射电镜观察细胞的形态学变化;流式细胞仪(AnnexinV-FITC)检测细胞凋亡率的变化;Western blot检测凋亡相关蛋白Bcl-2、Bax、Caspase-3、Caspase-9蛋白的表达情况。结果与对照组相比,蓝萼甲素对HeLa细胞有增殖抑制作用,呈剂量、时间依赖趋势;Bcl-2蛋白表达下降,Bax蛋白表达上调,Caspase-3、Caspase-9蛋白表达上调。结论蓝萼甲素对宫颈癌HeLa细胞有增殖抑制及诱导凋亡的作用,其分子机制可能与线粒体信号通路有关。     

维泰醇对小鼠淋巴白血病细胞促进凋亡的作用及其机制

维泰醇(alternol)是利用红豆杉树皮中一种微生物菌诱变株,经过发酵、纯化等工艺分离出的新型单体化合物。本研究探讨维泰醇对小鼠淋巴白血病(L1210细胞)的作用及其机制。采用MTT法检测维泰醇对细胞活力的影响，用形态学方法、 DNA凝胶电泳和流式细胞仪检测细胞凋亡，以Western blotting法检测与凋亡相关蛋白的表达。维泰醇对L1210细胞的增殖有明显抑制作用；处理后的细胞表现出凋亡特有形态学改变，细胞凋亡比率的增加有时间依赖性；维泰醇能降低细胞线粒体跨膜电位(ΔΨm)，增加细胞内活性氧(ROS)的水平，并引起细胞DNA发生片段化，形成典型的梯状条带；维泰醇能下调Bcl-2/Bax表达比率，并且上调caspase-3和caspase-9的表达，但是对caspase-8的表达没有明显的影响。维泰醇可能通过激活线粒体调控的凋亡通路诱导L1210细胞凋亡。     

Capsaicin induced apoptosis of B16-F10 melanoma cells through down-regulation of Bcl-2.

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), a pungent ingredient of hot chili peppers, has been reported to possess substantial anticarcinogenic and antimutagenic activities. In the present study, we investigated the effect of capsaicin on induction of apoptosis in highly metastatic B16-F10 murine melanoma cells. Capsaicin inhibited growth of B16-F10 cells in a concentration-dependent manner. Proapoptotic effect of capsaicin was evidenced by nuclear condensation, internucleosomal DNA fragmentation, in situ terminal nick-end labeling of fragmented DNA (TUNEL), and an increased sub G1 fraction. Treatment of B16-F10 cells with capsaicin caused release of mitochondrial cytochrome c, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase in a dose-dependent manner. Furthermore, Bcl-2 expression in the B16-F10 cells was slightly down-regulated by capsaicin treatment. In contrast, there were no alterations in the levels of Bax in capsaicin-treated cells. Collectively, these findings indicate that capsaicin-induces apoptosis of B16-F10 melanoma cells via down-regulation the Bcl-2.     

B16 and cloudman S91 mouse melanoma cells susceptibility to apoptosis after dacarbazine treatment

Considering the necessity of an individual choice of cytostatic drugs for patients with cancer disease and tumor cells' resistance to these compounds, their ability to induction of apoptosis should be investigated. The aim of this study was to determine the influence of dacarbazine (DTIC) on morphology and kinetics of proliferation of B16 and Cloudman S91 cells. It is important to determine the kind of death induced by the DTIC and the effect of a specific concentration. The evaluation of apoptosis and necrosis in these two mouse melanoma cell lines in vitro was performed. Induction of apoptosis was estimated in annexin V binding assay by flow cytometry. DNA content and cell cycle phases were determined by propidium iodide staining. DTIC induced morphological changes typical for apoptosis and necrosis in both cell lines. DTIC caused cell cycle arrest in S and G2/M phase of both cell lines which showed hypertetraploidy. The highest induction of apoptosis was observed in DTIC concentration of 200 microg/mL for B16 cells (11%) and 100 microg/mL for apoptosis Cloudman S91 cells (22.2%). Higher doses of DTIC caused intensification of necrotic process. The B16 melanoma cells are more sensitive to DTIC than the Cloudman S91 cells, however more intensive apoptotic process was detected in Cloudman S91 cells already at lower concentration of DTIC.     

大豆甙元对小鼠B16黑色素瘤细胞的分化诱导作用

大豆甙元在10～40μg·ml~(-1)浓度范围内,能够明显抑制B16细胞的增殖,受药物作用4d的B16细胞,克隆形成能力及体内成瘤能力明显降低,大豆甙元在抑制B16细胞增殖的同时,促进黑色素的生成,且对B16细胞形态具有明显的影响,低浓度时促使细胞平行排列,当浓度增加时,形成网状结构,黑色素颗粒明显增多。     

B16 and CLS91 mouse melanoma cells susceptibility to apoptosis after vincristin treatment in vitro

Tumor cells' chemoresistance is related to the occurrence or lack of apoptosis. Considering the individual choice of cytostatic drugs for cancer patients and tumor cell resistance the research was undertaken. The viability of mouse melanoma B16 and ClS91 cells and apoptosis induction in vitro after treatment with vincristin was examined. In the future, this kind of study may play an important role in the efficient choice of drug dose and in limiting the side-effects in patients treated with vincristin. Determination of vincristin's influence on cell proliferation kinetics, cell cycle progression based on DNA content and percentage of apoptosis and necrosis in B16 and ClS91 cells, was the object of the present study. The number of viable B16 and ClS91 cells was estimated by flow cytometry analysis. Apoptotic cells were detected using the annexin V-FITC test. Flow cytometric measurement allowed for simultaneous quantitation analysis of four cell subpopulations in the investigated probes. The subpopulations were viable, apoptotic, secondary necrotic and necrotic cells. After adding vincristin into B16 and ClS91 cultures, it was revealed that about 94% ClS91 cells and 45% B16 cells died in the apoptotic way. ClS91 melanoma cells were more sensitive to vincristin treatment than the B16 cell line. The EC50 value for the B16 line was 39.8 microM and for ClS91 was 16.7 microM. Cell cycle was established on the basis of DNA cell content after staining cells with propidium iodide and analysed by flow cytometry. Vincristin induced both B16 and ClS91 cell lines arrest in G2/M cell cycle phase. It was found a correlation between apoptosis occurrence in the melanoma cells and vincristin resistance.     

CLA-PTX对黑色素瘤B16-F10的体内外抗肿瘤作用

本研究旨在探索CLA-PTX对黑色素瘤B16-F10细胞的体内外抗肿瘤作用。选择鼠源B16-F10细胞系作为研究模型。研究CLA-PTX体外细胞毒,细胞凋亡,细胞周期作用,以及CLA-PTX体外细胞摄取作用。用荷瘤C57BL6/N小鼠研究CLA-PTX的体内抗肿瘤作用。体外细胞毒研究结果表明：CLA-PTX的IC50为（4.25±0.43）μM,优于紫杉醇（6.70±0.80）μM（P〈0.01）。与空白组和紫杉醇组相比,CLA-PTX可使细胞总凋亡比例增加（P〈0.01）。与空白对照组相比,CLA-PTX将细胞周期阻滞于S期,而紫杉醇则使细胞在G2‐M期蓄积。CLA-PTX的细胞摄取量显著高于紫杉醇（P〈0.01）。体内抗肿瘤药效显示,CLA-PTX抗肿瘤活性显著高于空白对照组和紫杉醇组（P〈0.01或P〈0.05）。上述结果表明,CLA-PTX对B16-F10细胞有显著体内外抗肿瘤作用。     

三氧化二砷诱导黑色素瘤B16细胞凋亡机制

目的 观察三氧化二砷（As2O3）对黑色素瘤B16细胞（B16细胞）的作用,探讨As2O3抗肿瘤的机制。方法 Hoechst33258染色观察细胞凋亡特征;应用激光扫描共聚焦显微术（LSCM）,结合特异性荧光探针Fluo-3／AM、H2EXEF-DA和DAF-FMDA,观察Asz03引起瘤细胞内钙离子（Ca^2＋）、活性氧（ROS）和一氧化氮（NO）的变化。结果 50μmol／L As2O3作用细胞12h后,Hoechst33258核染色可见典型凋亡细胞;细胞内Ca^2＋、ROS和NO含量明显高于对照组（P〈0．01）。结论 As2O3可诱导B16细胞发生凋亡,As2O3诱导凋亡可能与细胞内Ca^2＋、ROS和NO的增加有关。     

Combination of amino acids reduces pigmentation in B16F0 melanoma cells

Amino acids, the building blocks of proteins, play significant roles in numerous physiological events in mammals. As the effects of amino acids on melanogenesis have yet to be demonstrated, the present study was conducted to identify whether amino acids, in particular alanine, glycine, isoleucine and leucine, influence melanogenesis in B16F0 melanoma cells. Glycine and L-isoleucine, but not D-isoleucine, reduced melanogenesis in a concentration-dependent manner without any morphological changes in B16F0 melanoma cells. L-Alanine and L-leucine, but not D-alanine and D-leucine, also reduced melanogenesis without any morphological changes in B16F0 melanoma cells. However these amino acids did not show a concentration-dependency. Combination of L-alanine and the other amino acids, particularly 4 amino acids combination, had an additive effect on the inhibition of melanogenesis compared with single treatment of L-alanine. None of the amino acids affected the activity of tyrosinase, a key enzyme in melanogenesis. These results suggest that L-alanine, glycine, L-isoleucine and L-leucine, but not the D-form amino acids, have a hypopigmenting effect in B16F0 melanoma cells, and that these effects are not due to the inhibition of tyrosinase activity. Combination of these 4 amino acids had the additive effect on hypopigmentation that was as similar as that of kojic acid.     

23-羟基桦木酸对B_(16)细胞系的诱导分化作用

目的评价 2 3 羟基桦木酸对黑色素瘤B16细胞的抑瘤作用。方法以MTT法测定细胞增殖的抑瘤率 ,并以B16细胞形态、黑色素含量、细胞周期变化及体内致瘤能力的测定作为观察指标。结果用 10～ 2 0 μg/ml的2 3 羟基桦木酸作用肿瘤细胞 ,见有不同程度的抑瘤作用 (P <0 .0 0 1)。表现为黑色素生成能力增加 ,细胞生长缓慢。可使B16细胞阻断在G1期 ,肿瘤体积明显缩小。结论 2 3 羟基桦木酸低剂量 (10～ 2 0 μg/ml)对B16细胞有明显的分化诱导作用 ,而对体内外黑色素瘤增殖有明显的抑制作用     

In vitro and in vivo transfection of melanoma cells B16-F10 mediated by cholesterol-based cationic liposomes

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.