赫赛汀诱导乳腺癌细胞凋亡及对其细胞周期的影响

目的研究免疫靶向治疗药物赫赛汀(Herceptin)对HER-2过表达的乳腺癌细胞凋亡及细胞周期的影响。方法Herceptin处理体外培养的乳腺癌SKBR3细胞系,经MTT试验筛选最佳药物处理浓度和时间的组合,应用荧光显微镜、激光共聚焦显微镜、扫描电镜、透射电镜及流式细胞仪检测乳腺癌细胞凋亡的特征、凋亡细胞百分率及细胞周期的变化。结果在Herceptin作用下,荧光显微镜、激光共聚焦显微镜、扫描电镜、透射电镜观察SKBR3细胞均出现凋亡特征。Annexin/PI染色流式仪测定,药物处理组早期凋亡百分率较对照组显著增加(P<0.01)。流式仪细胞周期分析显示,S期细胞含量下降,而G2期细胞含量上升。结论免疫靶向治疗药物Herceptin可特异性地诱导HER-2过表达的乳腺癌细胞发生凋亡,并可使其生长受阻于G2期。诱导凋亡可能是Herceptin抗肿瘤作用的重要机制之一。     

A differential assay of NK-cell-mediated cytotoxicity in K562 cells revealing three sequential membrane impairment steps using three-color flow-cytometry

Annexin V and propidium iodide (PI) staining is a general technique for detecting apoptosis by flow-cytometry (FCM). The release of 2',7'-bis-(2-carboxyethyl)-5- (and-6)-carboxyfluorescein (BCECF), a non-lipophilic membrane-impermeable labeling dye, from the cytoplasm of target cells is an indicator of increased membrane permeability. This study aimed to devise a three-color FCM technique involving the BCECF-release parameter in addition to conventional Annexin V and PI staining for the analysis of target K562 cells undergoing cytotoxic/apoptotic processes mediated by natural killer (NK) cells. The results demonstrated the following step-wise process of membrane impairment: (1) initiation of Annexin V staining accompanied by increasing forward scatter (FSC) before BCECF-release, indicating membrane impairment without permeabilization by necrosis; (2) BCECF-release with decreasing FSC before PI influx; and (3) PI staining with the lowest FSC state. Therefore, the early stage of cytotoxicity/apoptosis conventionally defined by the flow-cytometric criteria of Annexin V staining before PI staining could be sub-divided into two stages before and after BCECF-release. Annexin-V staining in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was also initiated without BCECF-release. Although the underlying mechanism of the transition process from stage 1 to stage 2 is still unknown, this FCM technique should be a useful tool for differential assays of target cells regarding the sequential processes of NK-induced cytotoxicity.     

香烟烟气提取物对NR8383细胞吞噬功能的影响

目的 探讨不同浓度香烟烟气提取物(CSE)对大鼠肺泡巨噬细胞(NR8383)吞噬功能的影响.方法 通过CCK-8法分析细胞比活力,Annexin-V FITC/PI双染法检测细胞凋亡以及CFSE荧光标记分析细胞的分裂增殖,来确定CSE对NR8383细胞的作用浓度和作用时间.收集经不同浓度CSE处理24 h的NR8383细胞,与FITC标记的大肠杆菌共同孵育2 h后,用流式细胞仪检测胞内荧光强度,分析CSE对细胞吞噬功能的影响.结果 NR8383细胞的吞噬功能随着CSE浓度的变化先增强后降低.单独用CSE处理NR8383细胞,在100μg/ml CSE作用下,巨噬细胞的吞噬功能与正常对照组相比提高0.5倍,此时的细胞比活力最高.在CSE和LPS共同作用下,CSE的浓度为100μg/ml时,NR8383细胞的吞噬功能提高到正常水平的3倍.而当CSE浓度达到200μg/ml时,NR8383细胞的吞噬功能受到损伤,细胞凋亡率为54.1%.结论 CSE可以影响NR8383细胞的吞噬功能,在较低浓度时可能激活巨噬细胞的吞噬功能,但较高浓度时可能对巨噬细胞的吞噬功能造成损伤,CSE可能具有激活巨噬细胞吞噬功能的潜能.

Abstract：

Objective To investigate the phagocytosis function of cigarette smoke extracts (CSE)on the NR8383 cells. Methods The concentration of CSE and the optimal time was defined by cell counting kit-8 assay, Annexin V/PI cell apoptosis assay and CFSE cell proliferation assay. The cell was gained after exposed to the different concentration of CSE for 24 h and mixed with fluorescein-labeled Escherichia coli in 37℃ for 2 h. The fluorescence intensity was used to assay the phagocytosis function of NR8383 cells.Results The phagocytosis function of NR8383 cells may be changed by the concentration of CSE. In the concentration of 100 μg/ml, the phagocytosis function of NR8383 was enhanced 0.5 times than the normal cell when NR8383 cell was exposed to CSE, and the specific activity is the highest. When NR8383 cells were exposed to CSE and LPS, the phagocytosis function of NR8383 cells was enhanced 2 times than the normal cell. In the concentration of 200 μg/ml, the phagocytosis function of NR8383 cells was damaged, the rate of apoptosis is the 54. 1%. Conclusion Low concentration of CSE enhanced the phagocytosis function of NR8383 cells, but high concentration of CSE damaged the phagocytosis function of NR8383 cells. This study reveals a new role of CSE as an activator of macrophage function.     

抗人DR5单克隆抗体对人肝细胞系HL7702的凋亡作用

目的:探讨抗人DR5单克隆抗体(mDRA6)对人肝细胞系HL7702致凋亡的作用。方法:流式细胞术检测HL7702细胞表面DR5的表达。在荧光显微镜下观察mDRA6对HL7702细胞形态变化的影响;MTT法检测mDRA6的细胞毒性作用;AnnexinVFITC/PI双染法流式细胞术检测细胞凋亡率。结果:mDRA6导致HL7702细胞呈现典型细胞凋亡的形态特征;MTT法检测显示在40mg/L浓度下可杀伤39%的细胞;经流式细胞术检测显示,3mg/L的mDRA6作用HL7702细胞6h导致25.5%的细胞发生凋亡。结论:mDRA6能够诱导人肝细胞系HL7702凋亡,mDRA6具有诱导细胞凋亡的活性。     

流式细胞术定量检测细胞凋亡3种方法的比较研究

目的：探讨流式细胞术定量检测细胞凋亡３种方法的价值。方法：同时使用ＰＩ染色，ＴＵＮＥＬ及Ａｎｎｅｘｉｎ Ｖ／ＰＩ３壹量检测地塞米松处理小鼠的胸腺细胞凋亡发生率。结果：ＰＩ染色，Ａｎｎｅｘｉｎ Ｖ／ＰＩ，ＴＵＮＥＬ３种方法凋亡检出率分别为２７．１９％，３２．２８％，５０．１７％，两者之间均有显著差异。     

Flow cytometric evaluation of a model for phagocytosis of cells undergoing apoptosis

Phagocyte recognition of cells undergoing apoptosis is a rapid, efficient way of removing unwanted cells from tissue. The uptake of apoptotic cells prevents the release of potentially toxic cell contents that might otherwise damage neighbouring cells and elicit an inflammatory response. The aim of this work was to evaluate a simple cell culture assay to study phagocytosis of cells undergoing apoptosis. Fluorescent negatively charged beads (1 microm) or fluorescently labelled apoptotic cells, derived from etoposide-treated human monocytes (U937), were co-incubated with J774 cells or human peripheral blood macrophages for 1 h. Flow cytometry (FCM) showed an efficient uptake of both beads and apoptotic bodies. Phagocytosis of apoptotic cells but not of beads was significantly inhibited when macrophages were pre-incubated with cytochalasin D, suggesting that an experimental system based on beads is not an appropriate model of phagocytosis of apoptotic cells.     

抗人DR5单抗致白血病U937细胞凋亡的作用机制

目的探讨鼠抗人DR5单克隆抗体(mDRA-6)对白血病细胞U937凋亡诱导作用及机制。方法 MTT法、Annexin V-FITC/PI双染流式细胞仪检测mDRA-6对U937细胞的生长抑制及凋亡诱导作用,Western blotting检测caspase8、10、3、9及Cytochome C在U937细胞凋亡过程中的表达及激活情况;选用caspase8、10、3抑制剂预处理U937细胞,观察是否抑制mDRA-6对U937细胞的凋亡诱导作用。结果MTT结果显示:10mg/L的mDRA-6作用U937细胞24h,细胞死亡率为61.09%,呈时间、浓度依赖性;流式细胞仪检测显示mDRA-6作用4h,细胞凋亡率为69.03%;Western blotting结果显示caspase10、3、9及Cytochome C均有活性片断表达,而caspase8无明显激活;caspase10和caspase3抑制剂部分抑制mDRA-6的凋亡诱导作用,caspase8抑制剂作用不明显。结论抗人DR5单克隆抗体mDRA-6通过死亡受体和线粒体途径诱导白血病U937细胞凋亡。     

胰岛素样生长因子1对β样淀粉蛋白引起的神经元凋亡和tau蛋白磷酸化的作用(英文)

本研究的目的是阐明胰岛素样生长因子1(IGF-1)对β样淀粉蛋白(Aβ)引起的神经元凋亡的保护作用,以及tau蛋白磷酸化的作用。用MTT(四甲基偶氮唑盐)方法检测细胞活性,用流式细胞学结合Annexin V-FITC和PI(碘化丙锭)双染的方法检测早期凋亡和晚期凋亡/坏死,用Hoechst 33342染色观察凋亡细胞形态学,用免疫细胞化学的方法检测tau蛋白磷酸化。IGF-1阻止了Aβ25-35引起的培养的大鼠海马神经元的毒性,MTT值显著增加,从54.51%增至61.8%,Hoechst 33342阳性细胞的百分比从30.77%减少到22.81%。Aβ25-35孵育使Annexin V单标记细胞(Annexin V+/PI-)以及Annexin V/PI双标记细胞(An-nexin V+/PI +)的百分比显著增加(分别为3.41%和19.47%),应用100 ng/ml的IGF-1可显著减少Annexin V单标记细胞和Annexin V/PI双标记细胞的百分比分别至2.98%和15.16%。Aβ25-35可增加tau蛋白磷酸化,AT8阳性细胞占41.84%,而IGF-1则可抑制这一效应。我们的结果表明IGF-1可保护神经元,降低Aβ的细胞毒性,减少早期和晚期凋亡/坏死细胞的比例,抑制tau蛋白磷酸化,这可能是IGF-1神经保护作用的细胞机制。     

芹菜素对小鼠T淋巴细胞体外增殖、细胞周期和凋亡的影响

目的:研究芹菜素(AP)对小鼠T 细胞体外增殖、细胞周期和凋亡的影响.方法:无菌分离小鼠淋巴结和胸腺细胞,MTT 法检测不同浓度(25、50、100、150、200 μmol/L)的AP 对多克隆刺激剂刀豆蛋白A(ConA)诱导的T 细胞增殖的影响,PI 染色结合流式细胞术(FCM)分析细胞周期的分布,Annexin V-FITC/PI 双染色结合FCM 检测AP 对T细胞凋亡的影响,以及AP与地塞米松(DEX)共同作用下T 细胞凋亡的变化,并用MTT 法检测该药物浓度对T细胞的毒性作用.结果:25～200 μmol/L AP对T细胞无药物毒性作用,并明显抑制ConA诱导的T 细胞增殖(P<0.01),使细胞周期停滞在G0/G1期,且呈剂量依赖关系;各浓度AP对T细胞凋亡均具有抑制作用,并明显抑制DEX诱导的T细胞凋亡(P<0.01),且呈剂量依赖关系.结论:在一定浓度范围内,AP 明显抑制ConA诱导的小鼠T细胞体外增殖,使细胞周期停滞G0/G1期,并明显抑制T 细胞凋亡.     

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。     

流式细胞术检测细胞凋亡比较研究

从三方面探讨检测细胞凋亡流式细胞术的方法.选用大鼠皮质神经元细胞,同时使用碘化丙锭(PI)An-nexin V/PI.JC-1法检测细胞凋亡率.结果显示三种方法的凋亡率分别是:2.60%,6.52%和18.56%,两两之间有显著性差异(P＜0.01).JC-1法最敏感,AV/PI次之,PI单染法不适合检测早期细胞凋亡.     

The role of apoptotic resistance in irradiated adult articular chondrocytes

There have so far been no studies on the apoptosis of adult articular chondrocytes after X-ray irradiation. The purpose of this study was to assess the apoptotic resistance of articular chondrocytes in X-ray radiation, in order to examine the possibility of irradiated allogenic chondrocyte implantation. Adult human chondrocytes of the non-degenerated cartilage group without X-ray irradiation did not show positive cells of Annexin V and PI staining in a 48 h culture. The Annexin V positive chondrocytes did not increase in a radiation dose dependent manner, and the PI positive cells were slightly increased at 30 Gy irradiation. In the degenerated cartilage group, the PI positive chondrocytes without irradiation were present, and both the Annexin V and PI positive chondrocytes increased in a radiation dose dependent manner. The Annexin V and PI positive staining of chondrocytes in the non-degenerated cartilage group was less than that of the degenerated cartilage group in the same dose of X-ray irradiation exposure. Loss of the mitochondrial membrane potential, revealed in an early stage of apoptosis, did not show in the irradiated chondrocytes of the non-degenerated cartilage, but were demonstrated in those of the degenerated cartilage. These results demonstrated that the non-degenerated chondrocytes of X-ray irradiation were highly resistant for apoptosis, and this knowledge could be applied to allogenic chondrocytes implantation.     

小檗碱对人卵巢癌细胞SKOV3 增殖及凋亡机制的研究

目的:探讨小檗碱对人卵巢癌细胞(SKOV3)增殖及凋亡的影响。方法:MTT 法检测细胞增殖;流式细胞仪Annexin V/ PI 双染色法和透射电子显微镜检测细胞凋亡情况;甲基化特异性PCR 分析hMLH1 基因启动子区CpG 岛的甲基化状态;实时荧光定量RT-PCR 检测Bcl-2、Bax、Survivin 和hMLH1 mRNA 基因的表达。结果:小檗碱对卵巢癌SKOV3 细胞增殖有明显的抑制作用(P<0.05),呈剂量和时间依赖性。当与顺铂联用时,小檗碱对卵巢癌细胞有协同抗癌作用。小檗碱可明显诱导SKOV3 细胞凋亡,并下调Bcl-2、Survivin 基因及上调Bax 基因的表达。此外,小檗碱能恢复hMLH1 启动子的甲基化状态及增强hMLH1 mRNA 的表达。结论:小檗碱可抑制卵巢癌细胞增殖及诱导细胞凋亡,小檗碱可协同增强抗癌药物顺铂的抗肿瘤作用。     

抗DR5单抗增强顺铂诱导HeLa细胞凋亡作用的研究

肿瘤坏死因子相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)主要通过死亡受体5(death recep- tor,DR5)诱导肿瘤细胞凋亡。本文旨在探讨顺铂对HeLa细胞表面DR5分子表达影响及抗DR5单抗mDRA-6对顺铂诱导HeLa细胞凋亡增强作用。用间接免疫荧光染色结合流式细胞术分析DR5分子表达;MTT法检测HeLa细胞毒作用;用AnnexinV/PI双染试剂盒检测细胞凋亡率;荧光显微镜观察凋亡细胞的形态学改变。结果:正常HeLa细胞表面DR5表达量为30.01%,顺铂不能上调HeLa细胞表面DR5表达;mDRA-6可以明显提高顺铂对HeLa细胞的细胞毒作用,且存在剂量效应关系。IC_(50)值约为12.5μg/ml。研究结果表明,mDRA-6能够明显增强顺铂对HeLa细胞的细胞毒及细胞凋亡作用。     

己糖激酶2通过抑制线粒体凋亡通路减少LPS引起的人肺上皮BEAS-2B细胞凋亡

目的:探讨脂多糖(LPS)诱导人正常肺上皮BEAS-2B细胞凋亡的分子机制,并对己糖激酶2(HK2)在该效应中的作用进行分析。方法:采用不同浓度的LPS作用于BEAS-2B细胞建立损伤模型,CCK-8实验检测细胞存活率; Hoechst 33342染色及Annexin V/PI双染法分析细胞凋亡水平;通过使用线粒体凋亡通路抑制剂或者外在凋亡通路抑制剂鉴定细胞凋亡通路;在BEAS-2B细胞中转染HK2过表达质粒以验证HK2对上述效应的影响。Western blot法确认HK2过表达效果;免疫荧光实验检测HK2亚细胞定位。结果:CCK-8实验结果显示,LPS以时间和剂量依赖性方式降低BEAS-2B细胞活力; Hoechst 33342染色结果表明,给予LPS处理后的BEAS-2B细胞核出现固缩和碎裂;同时Annexin V/PI双染实验结果表明,处于凋亡状态的细胞由2. 89%增加至42. 4%,细胞凋亡率明显升高(P 0. 05)。线粒体凋亡通路执行蛋白caspase-9特异性抑制剂可显著抑制细胞凋亡,而caspase-8抑制剂却无此效应。在BEAS-2B细胞的凋亡过程中伴随着HK2的表达下调,而HK2过表达可以有效阻止以上事件的发生。结论:己糖激酶2可通过抑制线粒体凋亡通路减少LPS引起的人肺上皮细胞凋亡。     

Annexin V联合PI染色法定量检测凋亡细胞

为评价ＡｎｎｅｘｉｎＶ／ＰＩ双参数法在凋亡检测中的价值,用ＰＩ染色法、ＴＵＮＥＬ法及双参数ＡｎｎｅｘｉｎＶ／ＰＩ法定量检测地塞米松处理小鼠胸腺细胞凋亡发生率。结果发现其凋亡检出率分别为ＰＩ法２７１９％,ＡｎｎｅｘｉｎＶ／ＰＩ法３２２８％,ＴＵＮＥＬ法５０１７％。三者之间均有显著差异（Ｐ＜００１）。但ＴＵＮＥＬ法不能将死亡细胞、凋亡细胞区分开来。ＡｎｎｅｘｉｎＶ／ＰＩ法可将正常细胞（ＡｎｎｅｘｉｎＶ ＰＩ ）、凋亡细胞（ＡｎｎｅｘｉｎＶ＋ＰＩ ）、死亡细胞（ＡｎｎｅｘｉｎＶ＋ＰＩ＋）分开来。ＡｎｎｅｘｉｎＶ／ＰＩ法能检出早期凋亡细胞且更为简单、灵敏、特异,是较为理想的凋亡定量检测方法。     

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。     

一种新的检测细胞凋亡的多参数流式细胞分析方法

建立了一种新的检测细胞凋亡的多参数流式细胞分析方法.HL60白血病细胞株经化疗药物足叶乙甙处理后,加AnnexinV (AV)-FITC/PI孵育双染,用多参数流式细胞术检测细胞凋亡.结果显示,凋亡细胞的百分比随诱导时间的延长而逐渐增多.表明AV-FITC/PT双染法既能对细胞膜表面特异蛋白染色,又同时检测细胞膜完整性.多参数流式细胞术是一种快速、简便又准确的检测细胞凋亡的方法.     

CD28协同刺激分子对T细胞受体介导的胸腺细胞亚群凋亡的影响

目的：分析anti-TCRαβmAb anti-CD28mAb诱导小鼠胸腺淋巴细胞不同亚群的凋亡及凋亡程度，分析CD28协同刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响。方法：新鲜分离胸腺细胞，加入anti-TCRαβmAb-anti-TCRαβmAb anti-CD28mAb等培养20h，进行多重染色，流式细胞仪分析。结果：与胸腺细胞自发凋亡的结果相比较；（1）双信号刺激可明显增加胸腺细胞凋亡的数目，尤其是CD4^ CD8^ 胸腺细胞的凋亡数目。（2）凋亡的CD4^ CD8^ 亚群，CD4^ CD8^-亚群细胞表面CD28的表达均增多。结论：CD28共刺激分子对TCR受体介导的胸腺细胞亚群凋亡的影响与细胞的成熟程度有关，CD28共刺激分子能明显增强不成熟皮质胸腺细胞的凋亡。     

Cytotoxic effect of bone cements in HL-60 cells: distinction between apoptosis and necrosis

Ten PMMA-based bone cements used in prosthetic surgery have been studied with respect to the induction of programmed cell death (i.e., apoptosis) in HL-60 cells, which are remarkably sensitive to various apoptotic stimuli. Annexin V binding and propidium iodide (PI) exclusion were the methods for detection of early apoptotic changes, while PI entry was considered as a marker of necrosis. Hoechst 33342 staining was used to detect DNA fragmentation and Alamar blue was applied to measure oxide-reduction activity of cells. The production of reactive oxygen species (ROS) related to cell damage was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. Under our experimental conditions, the cements tested, for the most part, were not toxic to leukemic cells at 4 and 24 h. After 24 h, three cements were able to induce cell death, with two eliciting both apoptosis and necrosis, and one cement acting mainly via apoptosis. Both processes of cell death are likely to be mediated by the production of oxygen-free radicals. These findings provide potential leads for investigation into the molecular mechanisms of cell death, which are responsible for tissue damage by cements and intolerance of cemented prostheses.