干扰素通过上调BAX蛋白表达诱导肝癌细胞凋亡的研究

[目的]探讨干扰素(IFN)体外诱导肝癌细胞凋亡及其相关基因表达的作用。[方法]不同浓度的干扰素(IFN)处理肝癌细胞系CBRH-7919细胞不同时间后,光学显微镜下观察其形态学改变,用琼脂糖凝胶电泳检测作用不同时间干扰素对其细胞凋亡的影响,利用细胞免疫组化技术检测不同作用时间下BAX蛋白表达量的变化。[结果]干扰素作用肝癌细胞后,可看到较为典型的细胞凋亡形态学变化:细胞体积缩小,变形,细胞膜完整;细胞核固缩,染色质凝集,核碎裂,染色质片段化,凋亡小体形成等。琼脂糖凝胶电泳测得随着干扰素浓度的增高,出现典型的DNA LADDER。随着时间的增加BAX蛋白表达量出现增加,尤以48 h较明显。[结论]IFN可以诱导肝癌细胞凋亡,可能是由于IFN上调了BAX蛋白的表达,从而增强了自身对肝癌细胞的凋亡作用。     

BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis

BAX and BAK are "multidomain" proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca2+]er) that results in decreased uptake of Ca2+ by mitochondria after Ca2+ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca2+ adenosine triphosphatase) corrected [Ca2+]er and mitochondrial Ca2+ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca2+ from intracellular stores (such as arachidonic acid, C2-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to "BH3-only" signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca2+ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.     

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.     

VDAC2 inhibits BAK activation and mitochondrial apoptosis

The multidomain proapoptotic molecules BAK or BAX are required to initiate the mitochondrial pathway of apoptosis. How cells maintain the potentially lethal proapoptotic effector BAK in a monomeric inactive conformation at mitochondria is unknown. In viable cells, we found BAK complexed with mitochondrial outer-membrane protein VDAC2, a VDAC isoform present in low abundance that interacts specifically with the inactive conformer of BAK. Cells deficient in VDAC2, but not cells lacking the more abundant VDAC1, exhibited enhanced BAK oligomerization and were more susceptible to apoptotic death. Conversely, overexpression of VDAC2 selectively prevented BAK activation and inhibited the mitochondrial apoptotic pathway. Death signals activate "BH3-only" molecules such as tBID, BIM, or BAD, which displace VDAC2 from BAK, enabling homo-oligomerization of BAK and apoptosis. Thus, VDAC2, an isoform restricted to mammals, regulates the activity of BAK and provides a connection between mitochondrial physiology and the core apoptotic pathway.     

西兰苔叶黄酮体外抗癌活性的研究

[目的]探讨西兰苔叶黄酮粗提物（EOF）的抗癌机制。[方法]通过四甲基偶氮唑盐（MTT）试验检测EOF对细胞生长的抑制率,并用流式细胞仪采用Annexi-V/PI双染法进行细胞凋亡检测,以探讨西兰苔叶EOF对癌细胞增殖的抑制作用及其诱导的癌细胞凋亡机制。[结果]西兰苔叶EOF对SW480、HepG2、Hela和A549这4种细胞的生长均表现出抑制作用,抑制率随药物浓度的增加而增大,呈明显的量效关系,其50%抑制浓度（IC50）值分别为88.14、99.65、104.43、79.77μg/ml,对SW480细胞的作用最为明显。通过Annexi-V/PI双染法流式细胞仪测得其浓度为30、60、80、120、160μg/ml时,诱导的SW480细胞的早期凋亡率分别为12.74%、16.41%、19.61%、28.28%和50.66%。[结论]西兰苔叶EOF是通过抑制增殖和诱导细胞凋亡来发挥对肿瘤的治疗作用的。     

BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program

Although the proteins BAX and BAK are required for initiation of apoptosis at the mitochondria, how BAX and BAK are activated remains unsettled. We provide in vivo evidence demonstrating an essential role of the proteins BID, BIM, and PUMA in activating BAX and BAK. Bid, Bim, and Puma triple-knockout mice showed the same developmental defects that are associated with deficiency of Bax and Bak, including persistent interdigital webs and imperforate vaginas. Genetic deletion of Bid, Bim, and Puma prevented the homo-oligomerization of BAX and BAK, and thereby cytochrome c-mediated activation of caspases in response to diverse death signals in neurons and T lymphocytes, despite the presence of other BH3-only molecules. Thus, many forms of apoptosis require direct activation of BAX and BAK at the mitochondria by a member of the BID, BIM, or PUMA family of proteins.     

斑蝥酸钠维生素B6注射液诱导人膀胱癌EJ细胞凋亡实验观察

目的观察斑蝥酸钠维生素B6注射液对体外培养的人膀胱癌EJ细胞增殖抑制作用和凋亡的影响。方法应用流式细胞仪、CCK-8法、荧光倒置显微镜分析斑蝥酸钠维生素B6注射液作用EJ细胞后的变化。结果斑蝥酸钠维生素B6注射液对EJ细胞有增殖抑制作用,实验出现凋亡带及凋亡峰。结论斑蝥酸钠维生素B6注射液对体外培养的EJ细胞生长有明显的抑制作用,可以诱导EJ细胞凋亡。     

Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha

Accumulation of misfolded protein in the endoplasmic reticulum (ER) triggers an adaptive stress response-termed the unfolded protein response (UPR)-mediated by the ER transmembrane protein kinase and endoribonuclease inositol-requiring enzyme-1alpha (IRE1alpha). We investigated UPR signaling events in mice in the absence of the proapoptotic BCL-2 family members BAX and BAK [double knockout (DKO)]. DKO mice responded abnormally to tunicamycin-induced ER stress in the liver, with extensive tissue damage and decreased expression of the IRE1 substrate X-box-binding protein 1 and its target genes. ER-stressed DKO cells showed deficient IRE1alpha signaling. BAX and BAK formed a protein complex with the cytosolic domain of IRE1alpha that was essential for IRE1alpha activation. Thus, BAX and BAK function at the ER membrane to activate IRE1alpha signaling and to provide a physical link between members of the core apoptotic pathway and the UPR.     

超声照射对人胚肾细胞凋亡的影响

目的了解诊断超声照射对体外培养的人胚肾（HEK-293）细胞凋亡的影响。方法 HEK-293细胞用腹部超声探头（3.5 MHz、MI 0.8）照射0、10、20、30 min,再作Hoechst 33258染色,观察细胞凋亡的变化。结果超声照射10 min后,细胞凋亡率与假照组比较差异无统计学意义（P〉0.05）;超声照射20 min后,细胞凋亡率明显高于假照组（P〈0.05）,其中以照射30 min较为显著（P〈0.01）。结论特定剂量的超声照射可诱导人胚肾细胞凋亡增加。     

茶氨酸衍生物茶溴香酰胺脂质体对人肺癌细胞 生长和迁移的抑制作用

为了探索研究茶氨酸衍生物茶溴香酰胺制备的脂质体(TBrC-L)对人非小细胞肺癌A549细胞生长和迁移的抑制作用与其分子机制,采用薄膜分散法制备了TBrC-L,通过MTT法检测不同浓度的TBrC-L对人肺癌A549细胞生长的抑制作用;使用流式细胞术检测TBrC-L对人肺癌A549细胞凋亡的诱导作用;利用Transwell chamber法观察TBrC-L对人肺癌A549细胞迁移作用的影响;应用蛋白质印迹法检测人肺癌A549细胞中与凋亡和生长密切相关蛋白的表达和药物可能的作用靶点。实验结果显示,TBrC-L对人肺癌A549细胞生长和迁移有显著的抑制作用,促进肿瘤细胞凋亡、抑制肿瘤细胞生长和迁移可能是其抗人肺癌作用的重要机制,其作用的分子机制涉及到抑制EGFR、VEGFR1、VEGFR~2和Met受体介导的Akt和NF-κB信号传导通路,本研究结果提示,TBrC-L具有应用于临床治疗和(或)辅助治疗人肺癌的潜力。     

巴西菇多糖对肝癌细胞株凋亡的影响及其机制

目的：研究巴西菇多糖诱导人肝癌细胞株(SMMC—7721)凋亡的作用及机制。方法：采用电镜、DNA电泳、免疫组化法，对SMMC—772l株细胞凋亡以及凋亡抑制基因bcl—2的表达进行检测。结果：SMMC—772l株细胞经巴西菇多糖处理后出现核固缩、膜起泡、凋亡小体形成．琼脂糖凝胶电泳呈现出典型凋亡DNA梯状带．细胞内bcl—2表达较对照组低。结论：巴西菇多糖能诱导SMMC—772l株细胞发生典型凋亡．其抑制细胞内bcl—2的表达，是诱发肿瘤细胞凋亡的机制之一。     

盘花垂头菊中两种新型倍半萜类化合物诱导人肝癌细胞SMMC-7721凋亡

[目的]为开发新型肿瘤治疗药物提供依据。[方法]通过单细胞电泳和流式细胞分析法等技术,研究2种从盘花垂头菊中分离的高含氧甜没药烯型倍半萜化合物(HOBS)对人肝癌细胞SMMC-7721的抑制作用。[结果]2种HOBS处理48 h后,SMMC-7721细胞体积变小,且出现膜泡化和凋亡小体。单细胞电泳试验表明SMMC-7721细胞经不同浓度HOBS处理后,DNA碎片在电场中呈彗星状向阳极移动,且彗星样尾迹随药物浓度的增大而增长。HOBS能以剂量依赖的方式诱导SMMC-7721细胞凋亡。当2种HOBS浓度为8μg/ml时,细胞凋亡率分别达36.7%和38.9%。凋亡率随药物浓度的增大而升高,与对照组相比有显著差异,并出现典型的凋亡峰。2种HOB可使细胞内[Ca2+]i水平升高。[结论]HOBS能诱导SMMC-7721细胞凋亡。     

紫丁香苷抗肿瘤活性筛选及作用机制研究

[目的]筛选对紫丁香苷较敏感的人肿瘤细胞系,研究其抗癌机理。[方法]采用四氮唑盐法(MTT法)测定药物的体外抑制作用,通过测定Caspase-3活性研究细胞凋亡通路在抗癌活性中的作用。[结果]在常见的15种人肿瘤细胞中,紫丁香苷的半数抑制浓度IC50低于100μg/m L的有人肝癌细胞HepG2以及人前列腺癌细胞PC-3,紫丁香苷可促进肿瘤细胞中细胞凋亡因子Caspase-3活性增加。[结论]人肝癌细胞HepG2以及人前列腺癌细胞PC-3对紫丁香苷较为敏感,紫丁香苷的抑制作用呈现出剂量依赖的特点;细胞凋亡途径在紫丁香苷的抗肿瘤活性中起着一定的作用。     

Growth Inhibitory Effects of Garlic Polysaccharide on Human HepG2 Cells

[Objective] The growth inhibitory effects of garlic polysaccharide (GPS) on human HepG2 cells were evaluated in this paper.[Method] HepG2 cells were treated with GPS for 48 h for morphology assay by transition electron microscope.Anti-proliferative effects with the same treatment for 24 hand 48 h were assayed by MTT method.Cell cycle distribution and apoptosis assay of treated cells were performed in flow cytometry.[Result] The results showed that GPS enhanced growth inhibitory effect on HepG2 cells in a time-and dose-dependent manner.PI (Propidium iodide)/Annexin V staining analyzed by FCM (flow cytometry) demonstrated that GPS has a cytotoxic effect on tumor cells.Cell cycle arrest of HepG2 treated with GPS occurred in G2 phase.[Conclusion] This study suggests thatGPS could exert an antitumor effect and could be used as a therapeutic agent for live cancer.     

口虾蛄乙酸乙酯提取物体对人肺癌A549细胞的抑制作用

目的观察口虾蛄乙酸乙酯提取物（EOS）对人肺癌A549细胞的抑制作用。方法观察不同浓度EOS对肺癌A549细胞生长曲线和集落形成能力的影响,荧光双染法检测细胞凋亡情况。结果 EOS明显抑制肺癌A549细胞的生长和集落形成,呈剂量-时间依赖性;EOS作用48 h后肺癌A549细胞生长延缓并萎缩,胞核固缩;荧光双染实验可见细胞凋亡增多。结论 EOS可明显抑制人肺癌A549细胞的生长和增殖,并具有诱导凋亡作用。     

海洋珍珠生物提取液对人宫颈癌Caski细胞增殖与凋亡作用的影响

[目的]探讨海洋珍珠生物提取液对宫颈癌Caski细胞株增殖和凋亡的影响。[方法]MTT法检测细胞增殖情况;流式细胞仪采用AnnexinV和PI双染检测细胞的凋亡率,PI单染法测定细胞周期;Hoechst33258/PI荧光染色法观测Caski细胞的形态学改变;RT-PCR法测定宫颈癌Caski细胞株内Bcl-2和Bax基因表达。[结果]海洋珍珠生物提取液在一定浓度范围内,以浓度依赖性和时间依赖性的方式抑制宫颈癌Caski细胞的生长（P〈0.05）.以0、6、30和60μg/ml海洋珍珠生物提取液处理细胞24h,Caski细胞早期凋亡率分别为7.6%,7.15%,18.5%和38.9%,晚期凋亡比例为0.45%、2.85%、8.55%和23.2%。荧光显微镜下细胞呈现典型的凋亡性改变;RT-PCR显示海洋珍珠生物提取液处理后宫颈癌Caski细胞内的Bcl-2mRNA表达降低,BaxmRNA表达升高。[结论]海洋珍珠生物提取液以浓度依赖性和时间依赖性的方式抑制宫颈癌Caski细胞增殖,并通过降低Bcl-2,升高BaxmRNA来诱导细胞凋亡。     

ROS对氯化镉诱导的人乳腺癌MCF-7细胞凋亡的影响

为研究活性氧(ROS)对氯化镉(CdCl2)诱导的人乳腺癌MCF-7细胞凋亡的影响,本文采用荧光探针DHR检测CdCl2诱导的MCF-7细胞内ROS的变化水平;分别采用MTT法和DNA Ladder法检测CdCl2对细胞的毒性作用和细胞DNA的凋亡情况;通过加入自由基清除剂LNAC抑制细胞内的ROS水平,从而研究ROS对细胞凋亡的影响。结果显示,1.00×10-3 mol/L的CdCl2处理MCF-7细胞6h,细胞内ROS水平为对照组的2.04倍,细胞存活率为26.54%,DNA出现片段化分解。用CdCl2+LNAC复合处理MCF-7细胞6h,细胞内ROS水平为对照组的1.25倍,细胞存活率为61.17%,DNA不出现凋亡条带。因此,由CdCl2所导致的MCF-7细胞凋亡和DNA片段化分解,与细胞内ROS水平的升高有关。     

Apaf-1 and caspase-9 in p53-dependent apoptosis and tumor inhibition

The ability of p53 to promote apoptosis in response to mitogenic oncogenes appears to be critical for its tumor suppressor function. Caspase-9 and its cofactor Apaf-1 were found to be essential downstream components of p53 in Myc-induced apoptosis. Like p53 null cells, mouse embryo fibroblast cells deficient in Apaf-1 and caspase-9, and expressing c-Myc, were resistant to apoptotic stimuli that mimic conditions in developing tumors. Inactivation of Apaf-1 or caspase-9 substituted for p53 loss in promoting the oncogenic transformation of Myc-expressing cells. These results imply a role for Apaf-1 and caspase-9 in controlling tumor development.     

Role of Calcium Ion in Apoptosis of MD Cancer Cells Induced by Arsenic Trioxide

In order to observe the role of calcium ion in apoptosis of MD cancer cells induced by arsenic trioxide, inhibition percentage was detected by MTT assay; morphology changes were examined by fluorescence microscope; apoptosis was examined by DNA Ladder; [Ca^2＋]i was investigated by spectrofluorimeter in vitro on MDCC-MSB 1 cells. The results showed that As2O3 inhibited the proliferation of MDCC-MSB1 cells in concentration dependent manner （P〈0.05 or P〈0.01）; typical apoptosis character was observed by fluorescence microscope; DNA Ladder was observed; the [Ca^2＋]i was elevated significantly after the treatment of As203 （P〈0.05 or P〈0.01） and showed a dose-dependent manner. It is concluded that the calcium may play an important role in apoptosis of MD cancer cells induced by arsenic trioxide.     

梁金菇多糖诱导白血病细胞株HL-60凋亡的研究

目的：观察梁金菇多糖对自血病细胞株HL-60凋亡的影响。方法：HL-60细胞与不同浓度的梁金菇多糖共同孵育5d后，采用荧光显微镜与透射电镜观察细胞形态及结构、DNA凝胶电泳观察“阶梯状”条带、流式细胞仪测定凋亡率。结果：细胞出现核碎裂、核溶解，凋亡小体形成等典型的凋亡形态学变化。结论；梁金菇多糖能诱导HL-60细胞发生典型凋亡。