U937

U937（通用4篇）

U937 篇1

去甲斑蝥素 (norcantharidin, NCTD) 是由呋喃与马来酐按照Diels - Alder 加成反应后催化、氢化制得的一种新化合物, 构型与斑蝥素相似, 仅是 1, 2位无甲基。去除斑蝥素的2个甲基后, 对泌尿系统的刺激基本消失, 大量的体内外研究表明, 去甲斑蝥素对HeLa细胞、HL60细胞、 K562 细胞等生长有抑制作用[1,2,3]。

本研究通过体外培养U937细胞, 观察去甲斑蝥素对U937细胞的生长抑制作用, 同时观察去甲斑蝥素对U937细胞凋亡相关基因蛋白表达的影响, 探讨其与U937细胞凋亡的关系。

1 材料与方法

1.1 药物

去甲斑蝥素, 由北京第四制药厂生产, 分子式为C10H12O4, 相对分子质量为 196.200, 使用时用生理盐水稀释至所需的药物浓度。

1.2 主要试剂

免疫检测试剂盒 (包括二抗即生物素化山羊抗兔、正常山羊血清封闭液、HRP标记链霉卵白素、DAB显色剂) 、打孔剂 (Triton X-100) 、一抗 (兔抗鼠cFLIP抗体) 、四甲基偶氮唑盐 (MTT) , 均购自吉林市博蕴生物科技有限公司。

1.3 细胞株的培养和传代

将人白血病U937细胞接种于含10%灭活小牛血清的新鲜RPMI 1640培养液中, 于37 ℃、5%CO2培养箱中培养, 2～3 d传代1次 (用对数生长期的细胞进行试验) 。

1.4 MTT法检测细胞活性

参照参考文献[4], 取对数生长期U937细胞 (3×104/mL) 接种于96孔细胞培养板内, 200 μL/孔;每孔加入去甲斑蝥素20 μL, 使终浓度分别为5, 10, 20 nmol/L。每种浓度设3个平行孔, 另设阴性对照组。于37 ℃、5%CO2培养箱中培养44 h;离心, 弃上清液, 加无血清1640培养液200 μL及5 mg/mL的MTT 20 μL, 继续培养4 h;2 000 r/min离心5 min;弃上清液160 μL, 加入低渗盐水200 μL, 同上离心洗涤2次, 弃上清液200 μL;加入无水乙醇200 μL, 反复吹吸几次, 待蓝紫色结晶甲瓒 (formazan) 溶解后, 在酶标仪上于570 nm处读取OD值。

1.5 免疫组织化学法检测U937细胞凋亡相关基因蛋白的表达情况

1.5.1 细胞型FLICE抑制蛋白 (cFLIP) 表达量的测定

应用细胞涂片技术, 分组同1.3, 干预因素处理完毕后, 用冰丙酮固定5 min, PBS浸泡3 min;弃去上清液, 重复2次, 滴加3%H2O2, 室温静置5 min, 以去除内源性过氧化氢酶;PBS洗涤同前, 滴加0.1%TritonX-100 (打孔剂) 静置20 min;PBS洗涤同前;擦干组织周围的PBS液, 马上滴加20 μL正常山羊血清封闭液, 室温静置10～15 min;弃去上清液 (勿洗) , 用纸或纱布擦干组织周围的血清, 滴加20 μL一抗 (1∶100兔抗鼠cFLIP抗体) , 4 ℃过夜培养;洗涤同前;擦干组织周围的PBS后加入相应的生物素化的20 μL二抗 (1∶100生物素化山羊抗兔) , 室温静置10～15 min;洗涤同前;滴加20 μL 辣根过氧化物酶 (HRP) 标记的链霉卵白素, 室温静置10～15 min;洗涤同前;加20 μL DAB显色剂显色5～10 min, 镜下观察显色情况;将显色后的片子用自来水冲洗, 复染, 脱水, 透明, 封片。

参照参考文献[5], 每张免疫组织化学片子随机选取10视野, 采用HPIAS-1000高清晰度彩色病理图文分析系统, 选用阳性单位 (positive unit, PU) 指标定量表达免疫组织化学阳性反应。

1.5.2 存活蛋白 (survivin) 及胱天蛋白酶-3 (caspase-3) 表达量的测定

同cFLIP表达量的测定。

1.6 统计学分析

试验数据以平均数±标准差表示, 采用t检验分析试验数据, 检验水准为α=0.05。

2 结果 (见表1) 与分析

注:与阴性对照组相比, 同列数据肩标b表示差异显著 (P<0.05) , a表示差异极显著 (P<0.01) , 无肩标表示差异不显著 (P>0.05) 。

2.1 MTT法检测细胞活性

由表1可知, 与阴性对照组比较, 各浓度的去甲斑蝥素组细胞活性明显下降, 20 nmol/L去甲斑蝥素组细胞活性极显著下降 (P<0.01) 。

2.2 免疫组织化学法检测U937细胞凋亡相关基因蛋白的表达情况

免疫组织化学法检测发现, 阴性对照组胱天蛋白酶-3的PU值低于各浓度的去甲斑蝥素组, 特别是极显著低于20 nmol/L去甲斑蝥素组 (P<0.01) ;而阴性对照组cFLIP和存活蛋白的PU值高于各浓度的去甲斑蝥素组, 特别是极显著高于20 nmol/L去甲斑蝥素组 (P<0.01) 。说明去甲斑蝥素具有上调胱天蛋白酶-3表达, 下调cFLIP和存活蛋白表达的功能。

3 讨论

细胞凋亡受凋亡促进因子和凋亡抑制因子的共同调节, 尽管凋亡过程的详细机制尚不完全清楚, 但是已经确定胱天蛋白酶在凋亡过程中起必不可少的作用, 是细胞凋亡的核心机制, 其中胱天蛋白酶-3是细胞凋亡过程中重要的效应分子, 在细胞凋亡中起着不可替代的作用。存活蛋白是凋亡抑制蛋白IAP家族的新成员, 也是迄今为止发现的最强的凋亡抑制因子。cFLIP也是一种新发现的凋亡抑制因子, 它在多种肿瘤组织中表达增加[6], 对肿瘤的发生、发展起重要作用。cFLIP虽然有cFLIPL和cFLIPS 2种亚型, 但Irmler等人通过Western-blot分析发现, cFLIPL比cFLIPS具有更强的抑制Fas (factor associated suicide) 诱导凋亡的能力, 在分别转染了cFLIPS和cFLIPL的Jurkat细胞克隆中, cFLIPS的相对表达量高于cFLIPL, 但只有cFLIPL表达克隆可避免可溶性FasL (sFasL) 诱导的凋亡。因此, 本试验中只选择了cFLIPL作为检测指标。

本研究表明, 去甲斑蝥素对U937细胞的生长具有明显的抑制作用, 并且随着去甲斑蝥素浓度的增加细胞活性下降;同时去甲斑蝥素能上调胱天蛋白酶-3的表达, 下调cFLIP和存活蛋白的表达, 从而诱导U937细胞凋亡。

参考文献

[1]安巍巍, 王敏伟, 龚显峰, 等.去甲斑蝥素通过半胱氨酸天冬氨酸酶诱导HeLa细胞凋亡[J].中国病理生理杂志, 2005, 21 (3) :417-419.

[2]刘晓兰, 陈家旭, 刘燕, 等.去甲斑蝥素诱导HL60细胞凋亡的研究[J].北京中医药大学学报, 2000, 23 (4) :35-38.

[3]孙震晓, 魏育林, 赵天德, 等.斑蝥素及去甲斑蝥素诱导人红白血病K562细胞凋亡的细胞学研究[J].解剖学报, 2000, 31 (1) :56-59.

[4]赵良中, 时文艳, 鞠晓红, 等.玉竹提取物A对小鼠免疫性肝损伤治疗作用的研究[J].中国畜牧兽医, 2010, 37 (3) :160-163.

[5]申洪.免疫组织化学染色定量方法研究[J].中国组织化学与细胞化学杂志, 1995, 4 (1) :89-91.

[6]蒋月, 葛红, 叶柯, 等.Survivin和caspase-3在食管鳞癌术前放疗前后的表达与预后关系分析[J].中国肿瘤临床与康复, 2010, 17 (6) :481-483.

U937 篇2

热休克蛋白(heat shock protein,HSP)家族有多个成员,具有分子伴侣功能,能调节蛋白质的折叠、传递和降解,与细胞凋亡有着密切的联系。目前对HSP90的研究越来越受到重视。但是,关于HSP90在金葡菌诱导的U937凋亡过程中的作用尚未见报道。因此,我们设计实验,探讨HSP90表达与该凋亡过程的关系。

1 材料与方法

1.1 试剂

Monoclonal anti-β-actin antibodies抗体购自Santa Cruz生物技术公司(Santa Cruz,CA,USA),HSP90抗体购自Upstate公司(Charlottesvile,VA),An annexin V apoptosis凋亡试剂盒为RD公司产品;细胞培养试剂购自Invitrogen公司;其它所有试剂均购自Sigma公司(St.Lowis,MO)。

1.2 方法

1.2.1 准备细菌

金葡菌菌株Wood46由中山大学生命科学学院提供,根据文献[2]进行培养,用不含抗生素的含5%小牛血清的RPMI 1640培养基重悬并稀释。

1.2.2 细胞分离培养

人巨噬细胞系U937在含10%小牛血清、2 m M L-谷氨酸钠、100 U/mL青霉素和100 g/m L链霉素的RPMI 1640培养基中,于37℃、5%CO2的培养箱中培养。

1.2.3 金葡菌诱导U937凋亡

U937与金葡菌分别以0,1∶5,1∶10,1∶20,1∶50和1∶100的比例在37℃水浴培养30 min或当细胞:细菌为1∶20时分别培养0,15,30,60和90 min。然后离心2次(220g,8 min),洗去未吞噬细菌,置于含10%小牛血清、2倍青霉素及链霉素的RPMI 1640培养基中培养3h,获得感染的U937细胞。在一些实验中,金葡菌在60℃灭活30 min。

1.2.4 Annexin V FITC/PI双染分析

U937细胞的凋亡率用流式细胞仪检测。106细胞加入含有annexin V的60μL结合缓冲液,4℃避光15 min。为了区别早期凋亡和晚期凋亡或坏死细胞,细胞在检测前同时染annexin V和PI。细胞结合annexin V-FITC和PI后其结果采用CellQuest软件用流式细胞仪进行分析(FACS Calibur,BD Biosciences)。每个样品至少计数10 000个细胞。

1.2.5 Western blot

细菌感染细胞后用冰冷的PBS洗1次。包括1 m M Na2VO4和溶解缓冲液(50 m M Tris,pH 8.0,150 m M NaCl,5 m M EDTA,5%Glycerol,1%Triton X-100,25 m M NaF,2 m M Na2VO4,10μg/m L的各种抑肽酶、亮肽素和抑肽素)。细胞溶解产物被冰冻和融化3次,然后在4℃、14 000 g离心10 min。提取的细胞上清用DC蛋白分析试剂盒,用Lowry法测定总蛋白浓度(Bio-Rad,Hercules,CA)。按每孔加总蛋白50μg,进行SDS-PAGE凝胶电泳后,电转移至硝酸纤维素滤膜上(PVDF膜)。膜在包括0.05%Tween-20(TBST)的三羟甲基氨基甲烷缓冲液中用5%的脱脂奶封闭,然后加入兔抗人HSP90,4℃孵育过夜。应用β-Actin(1∶2 000)使蛋白量保持一致。然后用TBS-T缓冲液洗3次,加入辣根过氧化酶标记的羊抗兔IgG(Santa Cruz,CA),然后用化学发光法显色(PIERCE,Rockford,IL)。将滤膜用凝胶成像分析系统进行扫描,读取各条带的吸光度A值。

1.3 统计学分析

SPSS 10.0统计学软件进行统计学处理,凋亡率用均数±标准差统计。多组间均数比较采用单因素方差分析,组间两两比较用最小显著差法。当P<0.05时被认为具有统计学意义。每组实验均重复3次。

2 结果

2.1 金葡菌对U 937细胞凋亡的影响

U937细胞感染金葡菌后发生凋亡,并且凋亡百分率随着金葡菌浓度的增加及感染时间的延长而升高。1∶20,1∶50和1∶100组以及30,60和90 min组的细胞凋亡指数明显高于对照组(P<0.001),见图1。

2.2 金葡菌感染时H S P 90表达水平

我们用Western blot法检测了金葡菌感染是否通过HSP90通路。结果清楚地表明:HSP90的表达随着金葡菌浓度的增加及感染时间的延长而逐渐升高,见图2,图3。

3 讨论

高温、缺血和缺氧、化学毒物以及细菌或病毒感染等应激因子可以诱导细胞合成热应激蛋白,以适应或耐受不利的应激环境[3],故这类反应也被称为应激反应,热休克蛋白也被称为应激蛋白。热休克蛋白在应激条件下具有细胞保护作用[4]。热休克蛋白有多个家族,按其分子量大小主要分为HSP90、HSP70、HSP60和小HSP[5]。HSP90是一种分子伴侣,在保护蛋白质的正确空间结构、防止其意外降解和保护蛋白质功能方面有重要意义。HSP90的主要作用是结合已获得3级结构的蛋白质,参与这些蛋白质的激活与成熟过程[6]。研究表明,HSP家族是除Bcl-2之外的另一类抗凋亡基因家族。有关HSP90与凋亡的关系越来越受到重视。但是,关于HSP90在感染诱导的细胞凋亡过程中的作用,目前研究甚少。

许多病原体能够诱导细胞凋亡,包括大肠杆菌、金葡菌及结核杆菌等[7,8,9]。我们过去的研究发现:丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号转导通路中的三个成员P38MAPK,JNK和ERK在金葡菌诱导的U937细胞凋亡过程中起到了重要的作用[10]。在本研究中,我们应用金葡菌感染所致的凋亡U937细胞作为研究模型,探讨HSP90在这一过程中的作用。结果表明:U937细胞的凋亡率随着金葡菌浓度及感染时间的增加而逐渐升高,HSP90的表达随着金葡菌浓度及感染时间的增加而逐渐升高。

不同的细菌可能通过不同的信号转导通路诱导细胞凋亡,并且细胞凋亡信号转导途径的启动受细胞的来源、种类及生长环境等多种因素影响。关于金葡菌诱导U937细胞凋亡信号转导通路中的详细机理有待于进一步研究。HSP90表达与金葡菌所致的U937细胞凋亡的关系对于进一步认识金葡菌的致病机制及开发新型抗感染药物均具有一定的意义。我们以后的研究将进一步探讨在金葡菌诱导U937细胞凋亡过程中是否有其它信号转导通路的参与以及HSP90与这些信号转导系统之间的关系。

参考文献

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[4]NAKAJIMA M,KATO H,MIYAZAKI T,et al.Tumor immune systems in Esophageal cancer with special reference to heat-shock protein70and humoral immunity[J].Anticancer Res,2009,29(5):1595-1606.

[5]TAIYAB A,SREEDHAR AS,RAO CM.Hsp90inhibitors,GA and17AAG,lead to ER stress-induced apoptosis in rat histiocy-toma[J].Biochem Pharmacol,2009,78(2):142-152.

[6]POWERS MV,CLARKE PA,WORKMAN P.Death by chaper-one:HSP90,HSP70or both[J].Cell Cycle,2009,8(4):518-526.

[7]YOSHIDA A,INAGAWA H,KOHCHI C,et al.The role of Toll-like receptor2in survival strategies of mycobacterium tu-berculosis in macrophage phagosomes[J].Anticancer Res,2009,29(3):907-910.

[8]PARK WB,LEE JH,JEON JH,et al.Effect of tumor necrosis factor-alpha on intracellular Staphylococcus aureus in vascular endothelial cells[J].FEMS Immunol Med Microbiol,2008,52(2):247-252.

[9]ZHENG L,HE M,LONG M,et al.Pathogen-induced apoptotic neutrophils express heat shock proteins and elicit activation of human macrophages[J].J Immuno,2004,173(10):6319-6326.

U937 篇3

关键词：肿节风注射液 (SGI) ,U937细胞,细胞凋亡,共刺激分子CD80,共刺激分子CD86

肿节风 (Sarcandra glabra) , 别名草珊瑚、九节茶、九节风、接骨莲、抗肿I号等, 是金粟兰科 (Chloranthaceae) 植物草珊瑚 (Sarcandra glabra (Thunb.) Nakai) 的干燥全草[1], 是《中华人民共和国药典》2005年版一部收载的法定药材。其中挥发油、琥珀酸、延胡索酸、黄酮甙等是抗肿瘤有效成分。肿节风味苦、辛, 性平, 归心、肝经, 具有清热凉血、活血消斑、祛风通络之功效。临床用于血热紫斑、紫癜, 风湿痹痛, 跌打损伤。《陆川本草》曰:肿节风“破积, 止痛”;《闽东本草》言:肿节风“活血, 消肿胀”。现代药理研究表明, 肿节风具有镇痛、抗菌消炎、抗肿瘤、促进骨折愈合等多种活性[2]。

目前国内对肿节风的抗癌作用进行了较多的研究, 但其对白血病细胞增殖抑制作用的研究报道相对较少, 且其作用机制尚未清楚。因此本文通过体外培养用不同浓度的肿节风注射液 (SGI) 处理白血病U937细胞, 观察其增殖抑制作用, 以及共刺激分子CD80、CD86的表达, 探讨其作用机理, 为临床应用提供可靠理论依据。

1 材料与方法

1.1 药物

肿节风注射液:购自福建古田药业有限公司, 每支2m L (0.18g:4m L) , 批号:070513, 实验时用培养液稀释成所需浓度的无菌工作液。

1.2 细胞系及细胞培养

人单核细胞白血病细胞株U937:购自中国科学院上海细胞生物学研究所, 用含10%新生牛血清的RPMI-1640培养液培养, 每2~3天传代1次, 取对数生长期细胞进行实验。

1.3药物对U937细胞的增殖抑制作用

MTT法检测。

1.4不同浓度药物处理细胞后凋亡细胞的百分率检测

Annexin-V/PI双标法测定。

1.5 药物处理后细胞凋亡典型DNA梯形条带观察

DNA凝胶电泳分析。参考文献[3]方法, 不同浓度的SGI处理24、48、72小时后, 离心弃培养液, 收集1×106细胞, 经冷PBS洗涤后, 加入细胞裂解, 振荡, 收集上清, 沉淀物再重复提取1次, 合并上清, 加入终浓度为3μg/m LRNAase A和1%的SDS, 56℃水浴2小时, 然后加入终浓度为2μg/m L蛋白酶K, 37℃水浴2小时, 加入1/10体积的3M乙酸钠和2倍体积的冰无水乙醇, -20℃过夜沉淀DNA, 离心 (13000r/min, 25分钟) , 弃上清, 空气干燥。再加入TE缓冲液, 完全溶解后, 上样, 电泳2~3小时, 紫外光下观察并摄影。同样实验重复3次。

1.6 U937细胞经药物处理后共刺激分子CD80、CD86表达的检测

流式细胞术检测。

1.7药物处理后凋亡早、晚期细胞形态学变化和共刺激分子CD80、CD86表达的检测

激光共聚焦显微镜观察。

1.8 统计学处理

数据处理用均数±标准差 (±s) 表示, 两样本比较采用t检验, 以SPSS 17.0 for Windows软件包统计分析, 以P<0.05为差异显著的标准。

2 结果

2.1 不同浓度SGI对靶细胞 (U937) 的增殖抑制作用 见表1。

与空白组比较*P<0.05, **P<0.01

2.2 经药物处理后凋亡细胞阳性率的流式细胞术检测结果

Annexin-V/PI双标显示, 不同浓度的SGI处理细胞24小时未见明显的细胞凋亡, 与对照组相比, 差异不显著。当处理时间增至48小时, 细胞出现明显凋亡, Annexin-V+/PI-比例分别为2.2%、6.5%、14.1%、26.9%和37.8%, 而对照只有1.8%。72小时后, 随着细胞死亡增多, 相对凋亡比例有所下降, 作用不明显。见图1。

2.3 不同浓度药物处理后细胞凋亡出现典型的DNA梯形条带的DNA凝胶电泳分析

不同浓度的药物分别处理细胞48小时后, DNA凝胶电泳分析显示, 除了对照组和低浓度 (1.83g/L) 组未出现DNA梯形条带, 其他组均出现明显的DNA梯形条带 (如图2) 。提示SGI通过诱导细胞凋亡抑制细胞增殖。

2.4 凋亡早期和晚期细胞形态变化的激光共聚焦显微镜观察

将上述Annexin-V/PI双标过的细胞置于激光共聚显微镜下观察, 结果发现凋亡早期细胞因细胞膜保持完整, 通透性不高, 阻止PI进入细胞, 核无法染成红色, 视野内出现膜发绿色荧光, 核无色。当凋亡处于晚期时, 细胞濒临死亡, 膜仍完整但通透性增高, PI进入细胞, 将核染成红色, 则出现膜发绿色荧光, 核发红色荧光。

2.5 不同浓度SGI处理靶细胞后共刺激分子CD80、CD86表达

流式细胞术检测显示, 经不同浓度SGI处理靶细胞24小时后, CD80表达从对照组的3.0%升高到16.5%, 但随时间的延长, 表达未增加, 反而有所下降。当药物处理48小时后, CD86表达明显升高, 从对照组的1.1%升高至20.7%, 而后也有所下降, 这可能与细胞死亡增多有关。见图3~4。

2.6 经药物处理后共刺激分子CD80、CD86表达的激光共聚焦显微镜观察

将标记过CD80-FITC、CD86-PE细胞样品置于激光共聚焦显微镜下观察, 结果与流式细胞术检测CD80、CD86结果一致, 表达均有所增加。CD80-FITC在488nm波长的激发下, 发出绿色波长的荧光;而CD86-PE则发红色波长的荧光。见图5~6。

3 讨论

目前国内对肿节风的抗癌作用进行了较多的研究[4], 但其对白血病细胞增殖抑制作用的报道相对较少, 且其作用机制尚未清楚。本文通过观察不同浓度的SGI对U937细胞的增殖抑制作用发现低浓度 (1.83g/L) 对细胞作用不明显, 但随着浓度升高 (2.75g/L~7.32g/L) , 抑制作用也随之增强。Annexin-V/PI双标和DNA凝胶电泳均显示SGI能诱导细胞发生凋亡, 细胞凋亡率为2.2%~37.8%, 并出现典型的DNA梯形条带。激光共聚焦显微镜也观察到凋亡早期和晚期细胞的形态学变化。这些实验结果均提示SGI在一定浓度下能抑制白血病细胞增殖, 诱导白血病细胞凋亡。

T淋巴细胞是一种对肿瘤细胞有特异性杀伤作用的免疫细胞, 其特异性杀伤作用需通过表面的T细胞受体 (TCR) -CD4/CD8复合物识别肿瘤抗原-MHC复合体, 还需要B7-1 (CD80) 、B7-2 (CD86) 、ICAM-1、VCAM-1等共刺激分子间的连接传递第二信号 (也称为共刺激信号) 来完成完整的T细胞活化。Hirano N等[5]发现虽然白血病细胞能递呈白血病相关抗原肽, 但大多数白血病细胞表面不表达某些必要的共刺激分子如B7 (CD80、CD86) , CD80尤为缺乏[6], 使T细胞活化的第二信号不能有效传递, 从而不能有效地刺激T细胞分泌细胞因子, 诱导有效的抗白血病免疫。本文实验发现不同浓度的SGI能诱导共刺激分子CD80、86表达升高。处理U937细胞24小时后, CD80表达从对照组的3.0%升高到16.5%;当药物处理48小时后, CD86表达明显升高, 从对照组的1.1%升高至为20.7%。这些白血病细胞共刺激分子CD80、CD86表达增加, 在某种程度上可为T细胞完整活化提供必要条件。能否真正完整活化T淋巴细胞还需进一步检测共刺激分子 (CD80、CD86) mRNA和NFκβ转录因子表达的变化, 以及对混合淋巴细胞增殖和IFN-γmRNA表达的影响, 来确认其能否诱导有效的抗白血病免疫, 为临床应用提供可靠依据。

参考文献

[1]肖培根.新编中药志.北京:化学工业出版社, 2002:195.

[2]国家药典委员会.中国药典.北京:化学工业出版社, 2005:154.

[3]牛泱平, 高瑞兰, Helen Tao, 等.人参皂苷诱导HL-60细胞凋亡的研究.中国中西医结合杂志, 2002, 22 (6) :450.

[4]孙文娟, 李晶, 兰凤英, 等.肿节风注射液抗小鼠肝癌Hep-A-22的作用及毒性.中成药, 2003, 25 (4) :313.

[5]Hirano N, Takahashi T.Expression of costimulatory molecules in human leukemias.Leukemia, 1996, 10 (7) :1168.

U937 篇4

Several signaling pathways are known to regulate apoptotic processes,but the transcription factor,NF-κB,has a proapoptotic or antiapoptotic function depending on the cell type and stimulus[5].Heat shock proteins are known to inhibit various types of apoptosis.The HSP70 is the major inducible form of HSP in mammalian cells[6].However,the involvement of NF-κB and HSP70 in E.coli invasion of U937 cell lines has not yet been demonstrated.

1 Materials and methods

1.1 Materials

An Annexin V apoptosis detection kit was purchased from R D Systems(Abingdon,UK).Cell isolation and tissue culture reagents were obtained from Invitrogen Life Technologies(Lidingo,Sweden).AllotherreagentswereobtainedfromSigma-Aldrich(St.Lowis,MO).

1.2 Methods

1.2.1 Preparation of bacteria

E.coli strain ATCC25922(from College of Life Sciences,Zhongshan University)was cultured for 16 h and then washed and resuspended in RPMI 1640 medium containing 5%FCS.For heat inactivation,E.coli were incubated at 60℃for 30 min.

1.2.2 Cell culture

and treatment with E.coli U937 cells were used in all experiments,and they grow in RPMI 1640 medium supplemented with10%fetal calf serum,2 m M L-glutamine,100 u/mL penicillin,and 100 g/m L streptomycin(RPMI medium)incubating at 37℃in a humidified air containing 5%CO2.The medium was changed every 2days.Before infection with E.coli,the cells were washed,resuspended in RPMI medium without the antibiotics,and incubated in tissue-culture plates for 30 min at 37℃when U937∶E.coli was 0,1∶5,1∶10,1∶20,1∶50 and 1∶100 respectively,then cultured for 3 h in RPMI medium with the antibiotics.

1.2.3 Annexin V FITC/PI assay

U937 apoptosiswasquantifiedbyflowcytometryusing FITC-conjugated Annexin V and PI.Specific binding of Annexin V was achieved by incubating 106cells in 60μL of binding buffer saturated with Annexin V for 15 min at 4℃in the dark.To discriminate between early apoptosis and necrosis,the cells were simultaneously stained with Annexin V and PI before analysis.The binding of Annexin V-FITC and PI to the cells was measured by flow cytometry(FACS Calibur,BD Biosciences)using Cell Quest software.At least 10 000 cells were counted in each sample.

1.2.4 Isolation of nuclear proteins

Nuclear extracts were prepared as described previously[7].After treatment with curcumin for 24 h,cells were washed three times with phosphate-buffered saline and incubated on ice for 15 min in hypotonic buffer A(10 m M HEPES,pH 7.9,10 m M KCl,0.1 m M EDTA,0.1 m M EGTA,1 m M dithiothreitol,0.5 m M phenylmethylsulfonyl fluoride,and 0.6%Nonidet P-40).Cells were whirled gently for lysis,and nuclei were separated from the cytosol by centrifugation at 12 000 g for 1 min.Nuclei were resuspended in buffer B(20 m M HEPES,pH 7.9,25%glycerol,0.4 M Na Cl,1 m M EDTA,1 m M EGTA,1 m M dithiothreitol,0.5 m M phenylmethylsulfonyl fluoride)and shaken for 30 min at 4℃.Nuclear extracts were obtained by centrifugation at 12 000 g,and protein concentration was measured by Bradford assay(Bio-Rad,Richmond,CA).NF-κB in nuclear extracts was detected by Western blot as described below.

1.2.5 Western blot analysis

After treatment,briefly,cells were washed once with ice-cold phosphate buffered saline containing 1 m M Na2VO4and extracted with lysis buffer(50 m M Tris,pH 8.0,150 m M Na Cl,5 m M EDTA,5%glycerol,1%Triton X-100,25 m M Na F,2 m M Na2VO4,10μg/m L of each aprotinin,leupeptin and pepstatin).The cell lysate was frozen and thawed three times and were further centrifugated at 14 000×g for 10 min at 4℃to pellet insoluble material.The supernatant of cell extracts was analyzed for protein concentration by a DC protein assay kit based on the Lowry method[8].Equal amounts of protein(50μg)from each sample were separated on 10%sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes(MSI,Westborough,MA).Membranes wereblockedin5%nonfatdrymilkin Tris-buffered saline containing 0.05%Tween-20(TBST)and then incubated with HSP70(dilution1∶1 000)and NF-κB(dilution 1∶1 000)antibodies overnight at 4℃.Actin(dilution 1∶2 000)was used to control for equal protein loading.The immunoblots were then washed three times with TBS-T buffer,incubated with a horseradish peroxidase conjugated secondary antibody(goat anti-rabbit Ig M,Santa Cruz,CA),and developed using chemiluminescent substrate(PIERCE,Rockford,IL).To quantify and compare levels of proteins,the density of each band was measured by densitometry.

1.3 Statistical analysis

Each experiment was carried out in duplicate or triplicate and three or four independent experiments were performed.Results are expressed as means±standard deviation(SD)and analyzed with SPSS 11.5 software.Results were compared using analysis of variance(ANOVA).When ANOVA showedastatistically significantdifference,a group-by-group comparison was performed using a t-test with Tukey’s correction for multiple comparison.Statistical significance was set at P<0.05.

2 Results

2.1 U937 cell apoptosis induced by E.coli

In initial experiments,flow cytometry using FITC-conjugated Annexin V revealed that U937cells exposed to E.coli underwent apoptosis.This effect was positively correlated with the concentrations of E.coli,and excessive apoptosis was associated with loss of membrane integrity in an increased portion of U937,which indicates necrosis or late apoptosis(Figure 1).In contrast,apoptosis rate was similar to that of control if bacteria were heat-inactivated before the infection(Data not shown)

Note:1:Control;2～6:U937 infected by E.coli at 30 min for U937∶E.coli=1∶5,1∶10,1∶20,1∶50,1∶100 respectively.1)compared with control,P<0.05;2)compared with control,P<0.001

2.2 E.coli-induced apoptosis-related proteins HSP70 in U937 cells

To further examine the molecular mechanisms of E.coli,we focused on the expression of HSP70proteins.E.coli treatment significantly increased HSP70 production in a dose-dependent manner These experiments support the conclusion that E coli-induced apoptosis is mediated by HSP70 upregulation(Figure 2).

Note:1:Control;2～6:U937 infected by E.coli at 30 min for U937∶E.coli=1∶5,1∶10,1∶20,1∶50,1∶100 respectively

2.3 E.coli suppresses NF-κB activity

Since NF-κB is involved in both apoptotic and inflammatory responses,we examined whether the E.coli strain ATCC25922 could alter the NF-κB pathway.The data obtained demonstrated clearly that the expression of NF-κB decreased proportionally with increasing apoptosis(Figure 3).

Note:1:Control;2～6:U937 infected by E.coli at 30 min for U937∶E.coli=1∶5,1∶10,1∶20,1∶50,1∶100 respectively

3 Discussion

Pro-and antiapoptotic-signaling pathways strictly regulate the apoptotic process,and the relative balance between these opposing modulatory routes determines cell fate.Infection with E.coli stimulates numerous host cell-signaling pathways,which are known to be involved in cell death and/or survival.A recent study has shown that the probiotic E.coli strain Nissle 1917 induces gammadelta T cell apoptosis via caspase-andFas L-dependent pathways[9].We have shown that E.coli increase p38MAPK and JNK,decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937cells[10].However,the exact mechanism is still not fully understood.

Heat shock proteins(HSPs)are produced in cells in response to a range of stress-related stimuli,including heat,UV radiation,and microbial/viral infections[11].In addition to previously recognized activity of HSPs as facilitators of protein folding and chaperones,recent studies revealed unique properties of HSPs in generating specific immune responses against infectious agents.The down-regulation of HSP70 can facilitate the induction of apoptosis,whereas the up-regulation of HSP70 inhibits TNF-α-induced apoptosis.It was found that Helicobacter pylori infection significantly attenuated the expression of HSP70[12].It have shown that a link between pathogen-induced apoptotic neutrophils and up-regulation of the heat shock proteins HSP70.

The NF-κB family of transcription factors plays a critical role in numerous cellular processes.NF-κB is a transcriptional factor and its activation overrides the apoptotic response to Fas L or TNF,allows cell proliferation instead.However,in minor cases,the activation of NF-κB leads to apoptosis[13]The dissociation of the NF-κB/IκB complex is mediated by increased activity of the IκB kinase(IKK)complex.When organisms receive appropriate cues,the IKK complex becomes active,and it causes the phosphorylation of IκB.This phosphorylation leads to the ubiquitination and degradation of IκB,the release of NF-κB from its inhibitor,and its translocation from cytoplasm to the nucleus[14].Macrophage phagocytosis of M.tuberculosis is accompanied by activation of the transcription factor NF-κB[15].Deactivation of NF-κB pathways by YopJ is important for rapid Yersinia-induced apoptosis in macrophages.

Traditionally,HSP70 has been thought to protect cells by interfering with the mitochondrial apoptotic pathway;however,a growing body of evidence shows that heat shock or elevated HSP70can also promote receptor-mediated apoptosis[16].In particular,HSP70 can enhance cell death when it is overexpressed in cells also via inhibiting the NF-κB signaling cascade[17].It is suggested that elevated levels of HSP70 inhibit LPS-induced production of inflammatory cytokines by a mechanism involving inactivation of NF-κB[18].In this study,E.coli treatment significantly increased HSP70 production and decreased nuclear NF-κB activity in U937 cells.

Our investigation of the U937 cell death triggered by E.coli indicates a role of E.coli in the induction of apoptosis.We also showed that E.coli accelerates U937 cells apoptosis in a dose-dependent fashion.Additionally,our data showed that the expression of HSP70 is greatly upregulated and NF-κB downregulated in U937 cells.