电磁脉冲诱导肺癌细胞株A549凋亡的研究

目的　研究电磁脉冲 (EMP)对肺癌细胞A5 4 9凋亡的影响。方法　以场强为 6× 10 4V/m的EMP辐照 5次 /2min ,然后采用细胞计数、MTT、流式细胞术及免疫组化染色的图像分析 ,观察EMP对肺癌细胞A5 4 9的损伤作用 ,所有数据经SPSS8 0软件进行分析。结果　EMP可明显抑制肺癌细胞A5 4 9的增殖与活力。流式细胞术证明 ,A5 4 9细胞发生明显的凋亡。免疫组化的图像分析表明 ,伴有不同程度的Bcl 2蛋白表达的下调及P5 3蛋白表达的上调。结论　EMP可诱导肺癌细胞A5 4 9的凋亡。Bcl 2及P5 3蛋白参与了A5 4 9细胞的凋亡过程     

Induction of apoptosis by glyoxal in human embryonic lung epithelial cell line L132

Oxidative stress has been suggested to play a central role in the pathogenesis of lung fibrosis and lung epithelial cell apoptosis is considered to be a key event during fibrogenesis. Studies from various laboratories have indicated that metabolic conditions may initiate oxidative stress, thereby contributing to epithelial cell death. This study was designed to test the hypothesis that glyoxal, an intermediate product in the glycation reaction leading to advanced glycation end products (AGEs), may induce lung epithelial cell apoptosis. We investigated the in vitro effects of glyoxal on fetal human lung epithelial L132 cells. Immunocytochemical analysis of paraffin-embedded cells and fluorescence-activated cell sorter analysis revealed a dose-dependent accumulation of the glycoxidation product (epsilon)N-carboxymethyllysine (CML) in all compartments of the cell. It has been shown that CML modification of proteins may serve as an indicator for oxidative stress. To examine the role of apoptosis in epithelial lung cells we investigated glyoxal-dependent changes in pro- and antiapoptotic mediators bax and activated caspase-3, and galectin-3 and bcl-2, respectively. Increasing concentrations of glyoxal (50 to 400 microM) induced an increase in the number of apoptotic cells. The apoptotic changes were confirmed by transmission electron microscopy. Immunocytochemical analysis of treated cells revealed the presence of other AGEs such as pentosidine as well as products of lipid peroxidation.     

Induction of apoptosis by anti-CD44 antibody in human chondrosarcoma cell line SW1353

Because chondrosarcoma is resistant to chemotherapy and ionizing radiation, the primary treatment of chondrosarcoma is surgical resection. Effective chemotherapeutic agents for chondrosarcoma are necessary. Although there is evidence that CD44 is involved in apoptosis susceptibility in several cell types, the effectiveness of anti-CD44 treatment on chondrosarcoma has never been studied. This study was aimed to clarify whether anti-CD44 monoclonal antibody induces apoptosis in human chondrosarcoma cell line SW1353. Confocal microscopy revealed that the SW1353 cells expressed CD44 that bound the anti-CD44 antibody IM7. Treatment of the cells with IM7 resulted in a significant decrease in cell viability, compared with that with control IgG. In contrast, IM7 failed to reduce cell viability in human chondrocytes. In SW1353 cells, IM7 induced chromatin condensation, nuclear fragmentation, and apoptotic body formation while control IgG had marginal effect. These data indicate that anti-CD44 treatment could induce apoptosis in chondrosarcoma cells.     

JNK抑制剂对D-氨基葡萄糖衍生物诱导人食管癌Eca-109细胞周期阻滞和凋亡的影响

目的 观察特异性C-JUN氨基末端激酶(JNK)抑制剂SP600125对D-氨基葡萄糖衍生物2-(3-羧基-1-丙酰氨基)-2-脱氧-D-葡萄糖(COPADG)诱导的Eca-109细胞凋亡和细胞周期阻滞的影响,并探讨COPADG诱导Eca-109细胞凋亡的潜在分子机制.方法 体外培养Eca-109细胞,以COPADG及SP600125与细胞作用;Western blot法检测P-JNK蛋白表达,MTT法检测细胞增殖,流式细胞术检测细胞周期.结果 COPADG显著增加Eca-109细胞P-JNK蛋白的表达和细胞凋亡率,且诱导Eca-109细胞发生G0/G1期细胞阻滞,SP600125明显减少Eca-109细胞凋亡,并使G0/G1期细胞阻滞向G2/M期细胞阻滞发展.结论 COPADG可能通过激活JNK信号通路诱导Eca-109细胞凋亡.     

Induction of apoptosis by hexaminolevulinate-mediated photodynamic therapy in human colon carcinoma cell line 320DM.

Photodynamic therapy (PDT) typically involves systemic or topical administration of a tumor-localizing photosensitizer or prodrug and its subsequent activation by visible light. This results primarily in singlet oxygen-induced photodamage to the tumor. 5-Aminolevulinic acid (ALA) and its derivatives have recently been widely used for PDT due to their selective induction in tumor of endogenous protoporphyrin IX (PpIX), a potent photosensitizer. Although ALA-PDT has achieved successful results in the treatment of several clinical oncological and nononcological diseases, the mechanisms of this modality are still not fully elucidated. In the present study, the human colon carcinoma cell line 320DM was treated in vitro with PDT using hexaminolevulinate (HAL), a hexylester of ALA known to be 50 to 100 times more efficient at producing PpIX formation than ALA itself. PpIX production increased with increasing HAL concentrations in the cells and phototoxicity of the cells was enhanced with increasing light (450 nm) doses. HAL-PDT induced apoptotic cell death, as measured by nuclear staining of Hoechst 33342 for fluorescence microscopy, DNA electrophoresis and TdT staining for flow cytometry. PDT with 5 muM of HAL and a light dose of 640 mJ/cm2 produced a 75% apoptotic cell population 40 hr after the treatment. Furthermore, the loss of mitochondrial membrane potential coincident with the release of cytochrome c from the mitochondria into the cytosol led to a rapid activation of caspase-9 and caspase-3 (an executioner), indicating that the selective damage to the mitochondria by HAL-PDT can induce a cytochrome-c-mediated apoptotic response in the 320DM cells.     

双氢青蒿素诱导人前列腺癌细胞系PC-3凋亡

目的探讨双氢青蒿素(dihydroartemisinin,DHA)对人前列腺癌细胞系PC-3的凋亡诱导作用,并探讨其可能机制。方法人前列腺癌PC-3细胞经不同浓度(0、25、50和100μmol/L)DHA处理48 h,用FCM法检测各组细胞凋亡率。用荧光定量PCR检测细胞中HSP70 mRNA的表达。用蛋白质印迹法检测细胞中HSP70蛋白、凋亡酶激活因子(Apaf-1)及caspase-3的表达;荧光定量PCR及蛋白质印迹法增加两组,即100μmol/L HSP70抑制剂槲皮素(quercetin)作为阳性药物对照组,以DMSO作为溶剂对照组。结果 DHA能明显诱导PC-3细胞凋亡(P0.05)。不同浓度DHA能明显下调HSP70 mRNA及蛋白表达水平(P0.05),上调Apaf-1及caspase-3蛋白表达水平(P0.05)。结论双氢青蒿素能诱导前列腺癌PC-3细胞凋亡,其作用机制可能是DHA干扰HSP70的表达,促进caspase信号通路中Apaf-1及caspase-3表达。     

Analysis on the mechanism of Helicobacter pylori-induced apoptosis in gastric cancer cell line BGC-823

Helicobacter pylori (Hp) can induce apoptosis of gastric cancer cells. The mechanism of the process still needs further elucidating. This study was aimed to analyse the mechanism through which Hp induce apoptosis in human gastric cancer cell line BGC-823. The extract from VacA(+) and CagA(+) Helicobacter pylori strain NCTC11637 was applied to induce apoptosis. The expression, breakdown, and phosphorylation of proteins were probed by Western blotting with specific antibodies. Apoptosis of the cells was detected by flow cytometry. The results showed that incubating the cells with Hp extract caused the breakdown of both caspase-3 and -1. The breakdown was dose-dependent and correlated with the occurrence of the Hp extract-induced apoptosis. Among the substrates of caspase-3, DNA fragment factor (DFF) was degraded during incubation with Hp extract and a small fragment was released. However, poly(ADP-ribose) polymerase (PARP) did not break down during the incubation. Tyrosine kinase inhibitor Genistein prevented both the break down of caspase-3 and the apoptosis induced by Hp extract. MAPK/ERK inhibitor PD98059 did not prevent the apoptosis induced by Hp extract. The expression and activity of JNK, and the expression of Bcl-2 and Fas proteins did not change during the incubation with Hp extract. The results suggested that Hp extract initiated apoptosis in BGC-823 cells through activating tyrosine kinase, caspase-1, -3, and DFF.     

双氢青蒿素对人胰腺癌细胞系增殖和凋亡的影响

目的探讨双氢青蒿素对人胰腺癌细胞(PANC-1)增殖活性及凋亡的影响,及其作用机制。方法30、60、120、240和480μmol/L双氢青蒿素作用PANC-1 48 h后,采用MTT法检测双氢青蒿素对PANC-1增殖活性的影响,流式细胞术检测其细胞周期和凋亡的变化,Western blotting检测细胞内周期相关蛋白cyclin B1、Cdk1、p21及凋亡相关蛋白Bcl-2,Bax,Caspase-3,Caspase-9和细胞色素C(Cyto C)蛋白表达变化。结果 MTT实验结果表明,30~480μmol/L双氢青蒿素对PANC-1增殖活性具有明显的抑制作用(P<0.05)。流式细胞仪检测结果显示,双氢青蒿素可以使PANC-1细胞周期阻滞于G2/M期,并诱发PANC-1细胞发生凋亡,且随药物浓度呈现升高趋势(P<0.05)。Western blotting结果显示,双氢青蒿素作用后周期相关蛋白Cdk1和Cyclin B1蛋白表达降低,而P21蛋白表达升高。凋亡相关蛋白Bcl-2蛋白表达下调,Bax蛋白表达上调,Bax/Bcl-2蛋白表达比例升高,cleaved Caspase-3,cleaved Caspase-9和Cyto C蛋白表达升高。结论双氢青蒿素能抑制PANC-1增殖并诱发细胞凋亡,其凋亡机制可能与线粒体途径相关。     

赖氨大黄酸通过抑制EGFR信号通路诱导宫颈癌CaSki细胞凋亡

目的 研究赖氨大黄酸(RHL)对人宫颈癌CaSki细胞增殖、凋亡的影响及EGFR信号通路在其中的作用.方法 应用MTT法检测RHL对CaSki细胞增殖的影响;应用流式细胞仪检测细胞凋亡,应用Western blot检测EGFR信号通路蛋白表达水平及其蛋白磷酸化水平.结果 RHL能有效抑制宫颈癌CaSki细胞增殖,48和72 h的ⅠC50值分别是84.57 μmol/L和74.79μmol/L,RHL能诱导CaSki细胞凋亡,随药物浓度的增加,细胞凋亡率也逐渐升高;RHL能够抑制EGFR、AKT蛋白磷酸化,并显著下调NF-κB、BCL-2及Survivin蛋白的水平(P＜0.01),同时RHL还上调BAX蛋白的表达(P＜0.01).结论 RHL可能通过EGFR/AKT/NF-κB/Survivin信号通路诱导CaSki细胞凋亡.     

Induction of human IgE synthesis in B cells by a basophilic cell line, KU812

Induction of human IgE synthesis in B cells requires, in addition to IL-4 or IL-13, a second signal provided by CD40 ligand (CD40L) on activated Th2-type CD4⁺ T cells that do not or weakly express Fas ligand (FasL). Mast cells and basophils also produce IL-4 or IL-13 and express CD40L after immunologic or pharmacologic stimulation, although it is unknown whether these cells express FasL. This study investigated the capacity of KU812 cells, a human basophilic cell line, to produce IL-4 and IL-13, to express CD40L and FasL, and to induce IgE and IgG4 synthesis in human normal B cells. Upon stimulation of KU812 cells with either phorbol myristate acetate (PMA) or ionomycin (Iono), IL-4, but not IL-13, was produced in response to Iono, while IL-13, but not IL-4, was inducible by PMA. Moreover, both the time courses of IL-4 and IL-13 production and their amounts secreted were different; IL-4 production was transient, IL-13 production gradually increased, and IL-13 was heavily secreted as compared with IL-4. The combination of PMA and Iono (PMA/Iono) induced higher production of IL-4 or IL-13 than did Iono or PMA alone. KU812 cell-derived IL-4 and IL-13 had the ability to cause CD23 expression on B cells. PMA/Iono also up-regulated CD40L expression and induced a very low level expression of FasL. KU812 cells that had been activated by PMA/Iono followed by fixation could induce IgE and IgG4 synthesis in B cells in the presence of recombinant IL-4 or IL-13. This contact-dependent induction of IgE was completely abrogated by adding anti-CD40L MoAb or soluble CD40, whereas anti-FasL antibody did not significantly affect IgE production. These results indicate that activated KU812 cells produce biologically active IL-4 and IL-13, express functional CD40L, and exhibit weak induction of FasL, thereby supporting sufficient IgE production by B cells.     

白藜芦醇诱导人皮肤癌细胞系凋亡的可能信号通路

目的 探讨白藜芦醇(Res)诱导人皮肤癌细胞系(A431细胞)凋亡的可能信号分子,为临床相关疾病的治疗提供新的实验依据.方法 培养的A431细胞分为:对照组(A组)、Res(B组)、Res加SP600125(C组)以及单纯SP600125组(D组).免疫荧光染色、Western blot及相差显微镜观察等方法检测细胞形...     

内质网应激诱导人乳腺癌MDA-MB-231细胞系凋亡

目的探讨内质网应激(ERS)致雌激素受体阴性(ER-)人乳腺癌MDA-MB-231细胞系凋亡及钙激活中性蛋白酶2(calpain2)在凋亡效应中的作用。方法实验设空白对照组(DMEM)、对照组(DMEM+DMSO)和实验组,用不同浓度衣霉素(TM)诱导乳腺癌细胞不同时间,MTT法和流式细胞术检测细胞增殖抑制率及凋亡率;Western blot法检测葡萄糖调节蛋白78(GRP78)表达量,以GRP78表达最高点确定ERS模型建立,检测凋亡相关蛋白caspase4和CHOP表达以及calpain及其内源性抑制酶calpastatin的表达,用ERS抑制剂(4-PBA)和calpain抑制剂(calpeptin)分别预处理细胞2 h,观察对上述效应的影响。结果 9、12和18μmol/L TM诱导ERS对该细胞增殖有明显抑制效应,抑制率分别为33.88%±1.32%、51.51%±8.85%和55.77%±2.61%,细胞凋亡率分别为9μmol/L TM组16.70%±0.46%和12μmol/L TM组28.1%±1.09%,与对照组相比有明显差异(P0.05);9μmol/L TM诱导细胞24 h的GRP78表达上调最高(P0.01);ERS还可明显上调caspase4、CHOP和calpain表达,下调calpastatin表达(P0.01或P0.05),上述效应均能被4-PBA和calpeptin减弱或阻断(P0.05)。结论 ERS可诱导MDA-MB-231细胞凋亡,calpain2参与调控凋亡发生。     

高浓度花生四烯酸诱导小鼠成纤维细胞L929凋亡的研究

目的：探讨花生四烯酸是否能诱导小鼠成纤维细胞L929凋亡及其可能机制。方法：噻唑蓝（MTT）法检测细胞存活率,比色法测定乳酸脱氢酶（LDH）释放率,硫代巴比妥酸法测定细胞脂质过氧化产物丙二醛（MDA）含量,Hoechst 33258染色观察凋亡细胞核形态变化,DNA琼脂糖凝胶电泳检测DNA降解。结果：L929细胞在40-160 μmol/L花生四烯酸作用24 h后,细胞存活率明显下降,LDH释放率和细胞MDA含量显著增加（P<0.01）。经160 μmol/L花生四烯酸处理后的L929细胞呈现典型的凋亡核固缩表现。琼脂糖凝胶电泳显示DNA凋亡梯带。结论：高浓度花生四烯酸（80-160 μmol/L）能诱导小鼠成纤维细胞L929凋亡,可能与其促脂质过氧化作用有关。     

氯喹对正常胃上皮细胞GES-1 和胃癌细胞HGC-27 凋亡的不同影响

目的:比较氯喹对正常胃上皮细胞GES鄄1 和胃癌细胞HGC鄄27 凋亡的不同影响。方法:使用倒置显微镜观察CQ 处理后这两种细胞形态学变化;采用DAPI 核染色检测CQ 对HGC-27 细胞凋亡的作用;运用氯喹和雷帕霉素作用这两种细胞72 h 后,用CCK-8 检测这两种药物对两种细胞的增殖活性;JC-1 检测氯喹处理后细胞线粒体膜电位的变化;用免疫印迹法检测凋亡效应酶Caspase-3 和底物PARP 的改变。结果:10 滋mol/ L 的CQ 作用于GES鄄1 细胞和HGC鄄27 细胞72 h 后,在镜下可见,氯喹对GES-1 细胞的形态无明显影响,却能使HGC-27 细胞间隙增宽,悬浮细胞数目逐渐增多,细胞密度明显减少,细胞逐渐萎缩变圆,胞质减少,失去正常细胞形态;通过DAPI 核染色发现,氯喹作用两种细胞72 h 后,GES-1 细胞核浅染、核大小形态均未发生变化,HGC-27 细胞核呈浓缩致密的固缩形态或颗粒状荧光;CQ 和RAP 作用于正常胃上皮细胞和胃癌细胞HGC-27 72 h 后,CCK-8 结果显示相对于正常胃上皮GES鄄1 细胞而言,氯喹能抑制胃癌HGC鄄27 细胞的增殖活性;JC-1 结果显示氯喹作用于HGC鄄27 细胞后发生了红色荧光向绿色荧光的转变;Western blot 显示胃癌细胞HGC-27 的凋亡蛋白Caspase-3和PARP 表达显著减少。结论:相比正常胃上皮细胞GES-1,CQ 可以明显抑制人胃癌细胞HGC-27 细胞活力并诱导凋亡。     

PTPN13在人胃癌组织和胃癌SGC-7901细胞系中的表达及其对细胞增殖和侵袭的影响

目的分析PTPN13在人胃癌组织中及胃癌SGC-7901细胞系中的表达及其对细胞增殖、侵袭的影响。方法收集106例胃癌患者的胃癌组织及癌周正常组织标本。常规培养SGC-7901细胞并分为pc DNA3.1-PTPN13质粒转染组和未转染组。免疫组织化学法检测组织中的PTPN13表达;分析PTPN13表达与患者性别、年龄、肿瘤部位、肿瘤大小、浸润深度及转移的关系;Kaplan-Meier曲线分析不同PTPN13表达患者生存率的差异;CCK-8法检测细胞的增殖能力;Trans-well试验分析侵袭能力;Western blot检测E-cadherin、Snail及MMP9的表达。结果胃癌组织中的PTPN13阳性率低于癌周正常胃组织(31%vs 83%,P0.05);PTPN13的表达与肿瘤直径、浸润深度、淋巴结和远隔器官转移情况有关(P0.05);PTPN13阴性的胃癌患者2生存率较低;PTPN13过表达可以降低SGC-7901细胞的增殖率(P0.05),同时降低其侵袭能力(P0.05);上调PTPN13后SGC-7901细胞上皮化标志物E-cadherin表达增加,而间质化标志物Snail和MMP9表达减少。结论 PTPN13在胃癌组织和胃癌细胞中具有肿瘤抑制作用,较低的PTPN13表达提示患者预后不良。PTPN13具有成为胃癌的治疗诊断或治疗靶点的潜力。     

优化人胃癌细胞SGC-7901细胞骨架图像的激光共聚焦显微技术

目的利用激光共聚焦技术,对比三种固定液不同固定时间对胃癌细胞系SGC-7901的细胞骨架荧光染色的影响。方法选择三种常用固定液:2.5%戊二醛,4%多聚甲醛及95%乙醇,对接种24h后的SGC-7901细胞分别进行10min,20min及30min等不同时间点固定,5%BSA(含0.2%Triton X-100)封闭透膜1h,荧光抗体FITC标记的鬼笔环肽37℃避光孵育2h,DAPI室温染核15min,激光共聚焦扫描显微镜扫描,对比观察其染色结果。结果三种固定液及不同固定时间对SGC-7901细胞骨架染色效果不尽相同。95%乙醇固定后微丝之间区分不明,排列混乱,无网状结构,染色效果与固定时长无关。2.5%戊二醛固定后微丝结构完整,但层次不清晰。4%多聚甲醛固定后微丝结构完整,层次清晰,固定20min及30min微丝荧光染色清晰。结论 4%多聚甲醛固定20min对细胞骨架固定效果最好,荧光染色标记结果为后续细胞骨架功能研究优化实验方法并提供理论依据。     

PXD101诱导人前列腺癌细胞PC3凋亡的线粒体信号通路

目的:探讨PXD101(又称belinostat)诱导人前列腺癌PC3细胞凋亡的线粒体通路。方法:PXD101以不同刺激时间和剂量处理PC3细胞,CCK-8法检测细胞的活力;流式细胞术检测细胞的凋亡率和线粒体膜电位;Western blot检测线粒体凋亡相关蛋白Bcl-2、细胞色素C(Cyt C)和Bax;caspase-3活性检测试剂盒检测caspase-3活性。结果:PXD101能以时间和剂量依赖的方式抑制PC3细胞的存活(P0.05),流式细胞术检测结果表明PXD101处理后PC3细胞的凋亡率明显增加(P0.01)。PXD101能时间依赖性致线粒体膜电位降低和Bcl-2蛋白含量明显下降,Bax蛋白含量上升,促进线粒体释放Cyt C蛋白,caspase-3活性明显增强。结论:PXD101通过线粒体途径诱导人前列腺癌细胞系PC3细胞凋亡。     

bcl-3基因通过cyclin D1及Bax蛋白影响人结肠癌RKO细胞的凋亡

目的:研究bcl-3基因对人结肠癌RKO细胞迁移及凋亡的影响及机制。方法:采用人bcl-3基因的RNA干扰慢病毒载体沉默人结肠癌RKO细胞bcl-3基因的表达后,划痕实验观察bcl-3基因沉默前后RKO细胞迁移能力的变化,Annexin V/PI双染色法检测bcl-3基因沉默前后RKO细胞凋亡率的变化,Western blot法检测bcl-3基因沉默前后细胞周期蛋白cyclin D1及凋亡相关蛋白Bax、Bcl-2的变化。结果:划痕实验显示,划痕后36 h,bcl-3基因沉默前后RKO细胞划痕愈合率分别为84.00%及40.00%,差异具有统计学意义(P0.05)。Annexin V/PI双染色法流式细胞术分析显示,bcl-3基因沉默前后的RKO细胞均经5μmol/L顺铂处理24 h后,沉默前后的RKO细胞凋亡率分别为12.89%及59.67%,差异具有统计学意义(P0.05)。Western blot法检测显示bcl-3基因沉默后cyclin D1蛋白表达显著下降(P0.05),Bax蛋白表达显著上升(P0.05),但Bcl-2表达无明显变化(P0.05)。结论:沉默bcl-3基因后,RKO细胞迁移能力下降,凋亡率增加,并伴细胞周期蛋白cyclin D1及凋亡相关蛋白Bax表达的变化。bcl-3基因可能通过改变cyclin D1及Bax蛋白的表达而影响RKO细胞的凋亡。