槲寄生凝集素的制备与免疫学功能研究

目的:分析从中国槲寄生中提取的55 KD凝集素诱导T淋巴细胞增殖和肿瘤细胞凋亡的免疫学活性.方法:通过CM-Sepharose和ConA柱分离和纯化了分子量为55 kD的槲寄生凝集素.用流式细胞术和ELlSA检测纯化的槲寄生凝集素诱导T淋巴细胞表型和细胞因子分泌格局.并经Annexin V染色法研究其诱导肿瘤细胞凋亡的免疫学机制.结果:从中国槲寄生中分离纯化了由两个30 kD亚基组成的55 kD槲寄生凝集素蛋白,其具有特异诱导人γδT趼细胞扩增和以Caspase依赖途径诱导Jurkat细胞凋亡,并能促进TNF-α释放和抑制IL-10的释放的作用.结论:中国槲寄生凝集素具有免疫学调节作用和诱导肿瘤细胞凋亡作用.在研究抗肿瘤和免疫学机制中具有应用价值.     

甘露聚糖结合凝集素诱导人白血病细胞系U937细胞凋亡

目的研究甘露聚糖结合凝集素（MBL）对白血病细胞系U937凋亡的影响。方法不同浓度MBL处理U937细胞后,应用CCK-8法分析细胞增殖情况,Hoechst33258染色观察细胞核及染色质,AnnexinV/PI双染流式细胞术分析细胞凋亡率,real-timePCR和免疫印迹分析凋亡相关基因及蛋白表达。结果 30～50μg/mlMBL培养72h,U937细胞增殖明显受抑,出现不同程度核固缩、核碎裂;随MBL浓度升高或作用时间延长,凋亡细胞数逐渐增多;Fas、Caspase-3mRNA、Fas和FasL蛋白表达量升高,Caspase-3和多腺苷二磷酸多聚酶（PARP）蛋白被剪切激活或失活。结论 MBL可诱导白血病细胞U937细胞凋亡,其机制与上调Fas表达、剪切Caspase-3和PAPR有关。     

依达拉奉抗过氧化氢诱导血管内皮细胞凋亡作用的实验研究

目的探讨依达拉奉抗过氧化氢诱导血管内皮细胞凋亡作用及发生机制。方法采用0．5mmol／L浓度的过氧化氢作用于对数生长期的内皮细胞,建立细胞氧化损伤模型。在建模前采用依达拉奉进行干预,采用AnnexinV／PI染色后,进行流式细胞术检测内皮细胞的凋亡率,并检测caspase-3的水平表达与线粒体膜电位变化。结果在依达拉奉干预下,与过氧化氢组比较,高、中浓度组的早期凋亡阳性率、晚期凋亡阳性率、Caspase-3阳性率与线粒体损伤率等凋亡相关指标均明显降低,而低浓度组虽有减少趋势但无统计学意义。结论依达拉奉可以拮抗过氧化氢诱导血管内皮细胞凋亡的效应,估计与维持线粒体膜电位和减低caspase-3表达有关。     

基因转移及其诱导的肿瘤细胞凋亡

凋亡是一种特殊类型的细胞死亡，受某些基因及分子的调控。基因转移可导肿瘤细胞凋亡，根据不同肿瘤基因变异或基因表达的特点，采用不同的目的基因对肿效率细胞进行基因修饰以诱导细胞凋亡的发生可能成为肿瘤免疫基因治疗的有效手段。     

麦胚凝集素诱导小鼠成纤维细胞L929发生凋亡的研究

目的:研究麦胚凝集素(WGA)是否能够诱导小鼠成纤维细胞L929发生凋亡及其可能的分子机制。方法:分别收集以WGA或琥珀酰化WGA(sWGA)处理24 h的L929细胞,经碘化丙啶染色后,以流式细胞仪分析细胞凋亡百分率,同时以荧光显微镜观察吖啶橙染色细胞的形态。结果:WGA处理过的细胞,其DNA亚二倍体峰 (Sub-G₁) 即凋亡峰的百分率明显升高,且与WGA剂量呈正相关;而sWGA处理的细胞则无此现象。同时,荧光显微镜观察发现WGA处理的细胞发生核碎裂,但sWGA无此作用。结论: WGA能诱导L929细胞发生凋亡,而sWGA不能诱导该细胞凋亡,提示WGA结合至细胞表面唾液酸残基是其诱导凋亡所必需;WGA诱导细胞凋亡的能力可部分解释其细胞毒性。     

生物治疗诱导肿瘤细胞凋亡机制的研究进展

不同细胞毒性方法如化疗药物、放射线等通常是通过损伤DNA、激活p53依赖的凋亡途径而引起靶细胞的死亡,但往往同时也会对正常细胞造成损害或者引发二次恶变。生物治疗作为全新的肿瘤治疗模式,通过直接诱导肿瘤细胞凋亡或者抑制肿瘤的抗凋亡分子,其能够高度选择性地促进肿瘤细晌凋亡而不损伤币常细胞的DNA．因此具有很好的应用前景。     

抑制半乳糖凝集素3促进椎间盘软骨终板细胞凋亡诱导椎间盘退变

背景:椎间盘软骨终板的退变破坏了椎间盘完整性,影响了营养和代谢物交换及细胞外基质代谢平衡,是导致椎间盘退变的主要因素.半乳糖凝集素3(Galectin-3)是半乳糖凝集素家族的一员,参与调控细胞增殖、凋亡、细胞黏附等多种生理和病理过程,但是Galectin-3在椎间盘软骨终板中的调控作用尚不明确.目的:通过抑制剂GB1...     

survivin与肿瘤细胞凋亡

凋亡抑制蛋白 (ｉｎｈｉｂｉｔｏｒｏｆａｐｏｐｔｏｓｉｓｐｒｏｔｅｉｎ ,ＩＡＰ)是抑制细胞凋亡的重要成分。ｓｕｒｖｉｖｉｎ是最近新发现的一种ＩＡＰ家族成员之一。有证据表明 ,ｓｕｒｖｉｖｉｎ在大多数肿瘤中有表达 ,但在成人终末分化组织一般不表达或低表达。本文对ｓｕｒｖｉｖｉｎ的结构和功能、组织分布、抗凋亡的机制 ,以及与对恶性肿瘤诊治的价值作一综述。一、ｓｕｒｖｉｖｉｎ的结构和功能第一个ＩＡＰ蛋白是由Ｍｉｌｌｅｒ实验室首先在杆状病毒中发现的。以后又发现了一系列ＩＡＰ蛋白。ＩＡＰ家族蛋白包含 2个或 3个串联…     

藻蓝蛋白诱导喉癌HEP-2细胞凋亡的实验研究

目的:探讨藻蓝蛋白对人喉癌HEP-2细胞凋亡的影响,并初步探讨其机制。方法:不同浓度藻蓝蛋白处理HEP-2细胞,分别以MTT、倒置显微镜、扫描电镜、透射电镜、流式细胞术检测藻蓝蛋白对HEP-2细胞活力及凋亡的影响;DCFH-DA标记的流式细胞术检测细胞内的活性氧水平;分光光度法检测细胞内caspase-3、-8、-9的活性;RT-PCR、Western blot检测细胞凋亡相关基因的表达。结果:MTT结果显示藻蓝蛋白能够抑制喉癌HEP-2的细胞活力,且呈时间和剂量依赖性;倒置显微镜、电镜、流式细胞术的一系列定性化和定量化实验证实藻蓝蛋白可显著诱导HEP-2细胞的凋亡;藻蓝蛋白处理后细胞内ROS水平升高,caspase-3、-8、-9被激活;RT-PCR结果表明,藻蓝蛋白作用后Bax、Fas、P53、caspase-3和caspase-9表达显著上调(P0.05),Bcl-2表达显著下调(P0.05);Western blot结果和RT-PCR结果一致。结论:藻蓝蛋白能够诱导HEP-2细胞的凋亡,其机制可能与细胞内ROS水平升高,上调Bax、Fas和P53 mRNA,下调Bcl-2 mRNA的表达,从而促进凋亡信号的转导最终导致细胞凋亡有关。     

化疗药物诱导的肿瘤细胞凋亡和相关因子的调控

细胞凋亡是不同类型的细胞受到程序化因素控制而发生的细胞死亡过程 ,而这一过程在多个环节受多个因素调节。研究证明 ,细胞凋亡不仅与肿瘤的发生有关 ,而且与肿瘤细胞的耐药性关系密切。如果肿瘤细胞凋亡受到抑制 ,则表现为耐药。同样 ,通过各种方式促进肿瘤细胞凋亡的发生 ,则可增强肿瘤细胞的化疗敏感性。研究肿瘤化疗引起的细胞凋亡机制及其调控、对于指导临床化疗有重要意义。     

共固定化细胞因子诱导的HeLa细胞凋亡

利用光化学固定方法,将干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)共同偶联到高分子材料聚苯乙烯基板上,合成生物材料.从形态学、流式细胞仪定量、磷脂酰丝氨酸分析、Caspase活性四个方面进行研究.形态学和流式细胞仪研究结果显示,固定化(及游离)细胞因子都可以诱导HeLa细胞凋亡,而且发现游离细胞因子比共固定细胞因子发挥药效更快,但随着时间的增加,共固定化细胞因子的药效比游离的细胞因子更好、更持久有效.磷脂酰丝氨酸定量分析进一步证明了固定化细胞因子的长效活性.在Caspase-3活性研究中发现游离的药物处理后Capase-3活性的提高比共固定化药物处理后更明显,推测共固定细胞因子诱导的HeLa细胞的凋亡不仅存在Caspase依赖的通路,也可能存在caspase非依赖的通路.     

利拉鲁肽对缺氧诱导的心肌细胞凋亡蛋白Caspase-3和Bcl-2表达的影响

目的 检测胰高血糖素样肽-利拉鲁肽对氯化钴(cobalt chloride, CoCl2)诱导缺氧条件下的心肌细胞中凋亡蛋白Caspase-3和Bcl-2表达量的影响.方法 将原代培养的心肌细胞分为6组:空白对照组,CoCl2诱导化学缺氧组,缺氧+不同剂量利拉鲁肽处理组(1、10、100、1000nmol/L利拉鲁肽).结果 缺氧能诱导心肌细胞中凋亡蛋白Bcl-2表达的增高并抑制Caspase-3蛋白的表达(P<0.05);利拉鲁肽能抑制缺氧诱导的心肌细胞中凋亡蛋白Caspase-3的表达并增加Bcl-2的表达,且利拉鲁肽对凋亡蛋白的影响具有剂量依赖性.结论 利拉鲁肽抑制缺氧诱导的心肌细胞中凋亡蛋白Caspase-3的表达,并促进凋亡蛋白Bcl-2的表达.     

Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm². JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation.     

吗啡对培养新生大鼠心肌细胞凋亡影响的实验研究

目的利用体外原代培养的新生大鼠心肌细胞，研究吗啡对血清饥饿诱导的心肌细胞凋亡的影响。方法体外原代培养新生大鼠心肌细胞，分组给药后，用MTr法测心肌细胞的活力；用流式细胞仪分析心肌细胞周期；用Annexin V—FITC／PI双标记法测心肌细胞的凋亡率；用Westernblot法测Caspase-3蛋白的表达；用Fura-2双波长荧光法测心肌细胞内游离钙。结果体外原代培养的新生大鼠心肌细胞经无血清饥饿作用48h后，心肌细胞可出现明显的凋亡现象；给予不同浓度的吗啡作用于心肌细胞后，心肌细胞的这种凋亡现象可被抑制，其在1umol·L^-1处最为明显，表现为：心肌细胞凋亡率降低、Caspase-3蛋白表达减少、细胞内Ca^2＋离子浓度降低；给予10umol·L^-1纳洛酮（Naloxone）后，吗啡的这种抑制心肌细胞凋亡作用可被完全阻断；并且给予1umol·L^-1纳曲吲哚（Naltrindole）后，吗啡抑制心肌细胞凋亡的作用亦在一定程度上降低。结论吗啡可激活心肌细胞膜上的阿片受体，对血清饥饿诱导的心肌细胞凋亡具有抑制作用，且可被Naloxone阻断。     

Heat Shock Treatment Protects Human Hl-60 Cells from Apoptosis Induced by Lectin II Isolated from Korean Mistletoe,Viscum Album Var. Coloratvm

Abstract

Mistletoe lectin II (ML II) isolated from Korean mistletoe (Viscum album var. Coloratum), an effective therapeutic agent for cancers, is known to induce cell death via apoptosis. In the present study, we found the protective effect of heat shock treatment of human leukemia HL-60 cells against ML II-induced apoptosis. Exposure of HL-60 cells to ML II for 4 h resulted in apoptosis of the cells, which was evaluated by examining “DNA ladder” formation and DNA fragmentation assay. The DNA fragmentation was significantly reduced in the cells subjected to heat shock treatment by incubation at 42 °C for 1 h and subsequently allowed to recover for 2-16 h at 37 °C., prior to exposure to ML II. HL-60 cells transfected with heat shock protein (hsp) 70 gene exhibited resistance to ML II-induced apoptosis very similar to that seen when untransfected cells were heat-shocked. These results indicate that ML II-induced apoptosis in HL-60 cells is inhibited by heat shock treatment, at least in part, via a hsp 70-mediated mechanism.     

白藜芦醇诱导KG-1细胞凋亡作用机制的初步研究

通过观察白藜芦醇诱导人急性白血病细胞株KG-1凋亡的作用,检测bcl-2、caspase-3的表达,探讨白藜芦醇诱导白血病细胞凋亡的作用机制。采用噻唑蓝(MTT)比色法分析细胞生长状态;瑞-吉染色、透射电镜观察KG-1细胞凋亡的形态学变化;流式细胞术(FCM)测定细胞凋亡与周期分布;半定量RT-PCR检测bcl-2 mRNA、caspase-3 mRNA表达水平。结果显示白藜芦醇可明显抑制KG-1细胞增殖(P<0.01),与对照组相比,实验组使细胞发生S期阻滞(P<0.01),促进细胞凋亡(P<0.01),使bcl-2的表达下调,上调caspase-3的表达(P<0.05)。结论:白藜芦醇能诱导KG-1细胞凋亡,其机制可能与下调bcl-2、上调caspase-3表达水平有关。     

活血通脉汤对大鼠脑缺血再灌注损伤诱导的凋亡蛋白Fas、Caspase-3表达的影响

目的探讨活血通脉汤对大鼠脑缺血再灌注损伤过程中所诱导的凋亡蛋白Fas、Caspase-3表达的影响。方法制作脑缺血再灌注模型，80只大鼠随机平均分为4组，假手术组、模型组、阿司匹林组、活血通脉汤组，每组20只。应用免疫组织化学方法分别检测每组大鼠脑缺血再灌注12、24h脑组织Fas、Caspase-3表达的情况。结果模型组大鼠脑缺血再灌注12、24h脑组织Fas、Caspase-3表达水平明显高于假手术组（P〈0．01）；活血通脉汤组大鼠各时间点脑组织Fas、Caspase-3表达水平明显低于模型组（P〈0．05，P〈0．01），且与阿司匹林组无显著差异性（P〉0．05）。结论活血通脉汤能降低大鼠脑组织Fas、Caspase-3表达水平，减轻缺血脑组织神经元坏死的程度，对大鼠脑缺血再灌注损伤具有保护作用，机制与降低Fas、Caspase-3表达水平有关。     

Protein Kinase a or C Modulates the Apoptosis Induced by Lectin II Isolated from Korean Mistletoe,Viscum Album Var. Coloratum,in the Human Leukemic HL-60 Cells

Abstract

Mistletoe lectins (MLs) are increasingly used as an anticancer drug in the treatment of human tumors. The cytotoxic activity of MLs against tumor cells is due to programmed cell death (apoptosis). The up-or down-regulation of protein kinas A (PKA) or C (PKC) is known to be associated with the regulation of drug-induced apoptosis. Previously, we isolated cytotoxic MLII from the extract of Korean mistletoe (Viscum album var. Coloratum) and characterized its biochemical properties. The present study was designed to investigate the role of PKA and PKC in ML II-induced apoptosis. Exposure of human leukemia HL-60 cells to various doses of ML II resulted in apoptosis. However, the treatment of these cells with dibutyl-cyclic AMP (DB-cAMP), PKA activator, or 12-O-tertadecanoyl phorbol 13-acetate (TPA), PKC activator, suppressed ML II-induced apoptosis. Furthermore, KT5720 and staurospoline, PKA and PKC inhibitors, respectively, reversed the suppression by DB-cAMP and TPA in the ML II-induced apoptosis of HL-60 cells. These results     

Mechanism of Fibroblast‐Like Synoviocyte Apoptosis Induced by Recombinant Human Endostatin in Rats with Adjuvant Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by pronounced synovial hyperplasia, in which there may be an imbalance between the growth and death of fibroblast‐like synoviocytes (FLS). The present study was undertaken to examine the effect of recombinant human endostatin (rhEndostatin) on FLS apoptosis in experimental RA. Adjuvant arthritis (AA) was induced in male Sprague Dawley (SD) rats. Using cultured AA FLS obtained from these rats, the apoptosis process was measured by terminal deoxyribonucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) as well as Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide (PI) labeling methods. In addition, the expression levels of the Fas, c‐jun, NFκB, and caspase‐3 gene products in synovial tissues were quantified by quantitative real‐time polymerase chain reaction (qPCR) and/or Western blotting assays. Our data revealed that rhEndostatin induced apoptosis in AA FLS. The number and signal density of TUNEL‐positive cells were significantly increased in rats treated with rhEndostatin (2.5 mg/kg). The percentage of Annexin V‐FITC‐positive cells was 6.67% after treatment with rhEndostatin at 25 μg/mL for 48 hr, compared with only 3.32% among untreated control cells. There were significant increases in Fas mRNA, c‐jun mRNA, c‐Jun protein, and caspase‐3 (p20) protein in AA synovial tissues treated with rhEndostatin (2.5 mg/kg), whereas no significant difference in NFκB expression was detected between treated and untreated tissues. These findings indicate that rhEndostatin has a therapeutic effect on RA by inducing FLS apoptosis, which is strongly associated with increased expression of Fas, c‐jun, and caspase‐3, but not NFκB. Anat Rec, 291:1029–1037, 2008. © 2008 Wiley‐Liss, Inc.