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改良支气管肺泡灌洗液细胞分类计数制片及染色法

改良支气管肺泡灌洗液细胞分类计数制片及染色法徐佳;黄媛;吴卫;董玉香;崔京涛;崔巍【摘要】目的改良支气管肺泡灌洗液细胞分类制片及染色方法,并对其临床实用性进行验证.方法随机抽取50例门诊及住院患者肺泡灌洗液标本,分别用改良的自动化瑞氏吉姆萨染色法及传统手工HE染色法染色,由两名具有专业技术能力的检验人员分别进行显微镜下细胞分类计数,评估两种制片方法检测结果的可比性.结果两种方法对肺泡灌洗液中吞噬细胞、中性粒细胞、淋巴细胞和嗜酸性粒细胞计数一致性较好,相关系数(r)均＞0.95.改良法染色的细胞形态完整,细胞核和细胞浆分辨清楚,可见颗粒、空泡等细微结构;而传统法染色后的细胞固缩明显,细胞体积变小,细胞核和细胞浆分辨清晰度下降,颗粒和空泡等细胞结构辨认不清.改良法比传统法省去了滤液离心富集细胞的过程(6 min),并且使用自动化瑞氏吉姆萨染色法(20 min)代替了手工HE染色法(30 min),每份标本至少节省了15～20 min的制片和染色时间.结论改良的自动化瑞氏吉姆萨染色法优于手工HE染色法,更适用于支气管肺泡灌洗液细胞分类计数,尤其是批量检测.【期刊名称】《临床检验杂志》【年(卷),期】2014(032)002【总页数】4页(P98-101)【关键词】支气管肺泡灌洗液;细胞分类计数;瑞氏吉姆萨染色;HE染色【作者】徐佳;黄媛;吴卫;董玉香;崔京涛;崔巍【作者单位】北京协和医院检验科,北京100730;北京协和医院检验科,北京100730;北京协和医院检验科,北京100730;北京协和医院检验科,北京100730;北京协和医院检验科,北京100730;北京协和医院检验科,北京100730【正文语种】中文【中图分类】R446支气管肺泡灌洗(bronchoalveolar lavage，BAL)技术是一种通过纤维支气管镜对支气管以下肺段或亚肺段以无菌生理盐水反复灌洗、回收、获取样本，并进行检查与分析的技术[1]。

巨噬细胞极化对心肌成纤维细胞活化的影响

天津医药2020年7月第48卷第7期巨噬细胞极化对心肌成纤维细胞活化的影响吴惠娟1，张盛昔1，杨潇2，胡因铭1，王乐旬1△，郭姣1摘要：目的观察巨噬细胞极化上清对心肌成纤维细胞活化的影响。

方法提取SD 大鼠的骨髓细胞和心肌成纤维细胞。

利用巨噬细胞集落刺激因子（M-CSF ）处理骨髓细胞后，加入刺激因子：M0（无刺激因子）、M1（100μg/L 脂多糖+10μg/L 干扰素-γ）、M2（20μg/L 白细胞介素-4）诱导巨噬细胞极化。

将极化后的不同型别巨噬细胞及其培养上清分别与心肌成纤维细胞共培养，分别设空白对照组、M0组、M1组和M2组，通过细胞免疫荧光检测心肌成纤维细胞中纤维化蛋白的表达水平；实时荧光定量逆转录聚合酶链反应检测巨噬细胞和成纤维细胞特征分子的表达；Western blot 检测纤维化相关蛋白及转化生长因子β受体（TGFβR ）、血小板衍生生长因子受体（PDGFRs ）信号通路活化情况。

结果经M-CSF 及相应刺激因子诱导，成功获得M1和M2型巨噬细胞。

细胞共培养结果显示，与M0组相比，M1组上清培养的心肌成纤维细胞中胶原蛋白1（Col1a1）和Col3a1的mRNA 水平以及平滑肌肌动蛋白（α-SMA ）表达水平显著降低（P ＜0.05），而M2组上清培养的心肌成纤维细胞中Col1a1和Col3a1的mRNA 水平以及α-SMA 、结缔组织生长因子（CCN2）表达水平显著升高（P ＜0.05）。

M1组上清培养的心肌成纤维细胞中PDGFRβ蛋白磷酸化水平显著低于M0组（P ＜0.01），而M2组上清培养的心肌成纤维细胞中PDGFRβ蛋白磷酸化水平显著高于M0组（P ＜0.05）。

结论M1型巨噬细胞上清能够抑制心肌成纤维细胞活化，而M2型巨噬细胞上清能够激活心肌成纤维细胞。

M1型巨噬细胞抑制纤维化的作用可能与抑制PDGFRβ通路的活化有关。

关键词：纤维化；心脏；成纤维细胞；巨噬细胞；受体，血小板源生长因子β中图分类号：R392.12文献标志码：ADOI ：10.11958/20200169Effects of macrophage polarization on the activation of cardiac fibroblastWU Hui-juan 1,ZHANG Sheng-xi 1,YANG Xiao 2,HU Yin-ming 1,WANG Le-xun 1△,GUO Jiao 11Guangdong Metabolic Disease Research Center of Integrated Chinese and Western Medicine,Guangdong Key Laboratory of Metabolic Disease Prevention and Treatment of Traditional Chinese Medicine,Joint Laboratory of Guangdong,Hong Kong and Macao on Glycolipid Metabolic Diseases,Institute of Chinese Medicine Sciences,Guangdong Pharmaceutical University,Guangzhou 510006,China;2Department of Clinical Laboratory,Guangzhou First People's Hospital△Corresponding Author E-mail:*********************Abstract:ObjectiveTo observe the effects of macrophage polarization supernatant on the activation of cardiacfibroblasts.MethodsBone marrow cells and cardiac fibroblasts of SD rats were extracted.Bone marrow cells were inducedto M1and M2by treating with macrophage colony-stimulating factor (M-CSF),and cells were divided into M0group (no stimulating factor),M1group (100μg/L LPS+10μg/L INF-γ)and M2group (20μg/L IL-4).Different macrophages were co-cultured with cardiac fibroblasts,and different macrophage supernatants were collected to culture with cardiac fibroblasts.Immunofluorescence was performed to examine the fibrotic protein expression in cardiac fibroblasts.The mRNA levels of macrophage-specific molecules,fibrosis-related genes and signaling pathways were tested by real-time PCR.The fibrosis-related proteins and the activation of TGFβR and PDGFRs signal pathways were detected by Western blot assay.Results After treatment with M-CSF and stimulating factors,M1macrophages and M2macrophages were pared with the M0group,the mRNA levels of Col1a1and Col3a1and the protein level of α-SMA were significantly decreased in the cardiac fibroblasts treated by the supernatant of M1macrophage group (P ＜0.05),while the mRNA levels of Col1a1and基金项目：广东省自然科学基金博士启动纵向协同项目（2018A030310403）；广东省自然科学基金（2018A0303130168）；广东省医学科学技术研究基金（A2018068）；广东省基础与应用基础研究基金项目（2020A1515010155）作者单位：1广东省代谢病中西医结合研究中心，广东省代谢性疾病中医药防治重点实验室，粤港澳联合代谢病重点实验室，广东药科大学中医药研究院（邮编510006）；2广州市第一人民医院检验科作者简介：吴惠娟（1995），女，硕士在读，主要从事中医药防治糖脂代谢病方面研究△通信作者E-mail ：*********************细胞与分子生物学611Tianjin Med J,July2020,Vol.48No.7《中国心血管病报告2018》显示我国心血管病现患人数约为2.9亿，其中90%以上与心脏有关［1］。

肿瘤动物模型的构建——白血病篇知识讲解

肿瘤动物模型的构建——白血病篇肿瘤动物模型的构建——白血病篇导读白血病（Leukemia）是一种常见的恶性血液疾病，俗称血癌。

据统计，白血病是儿童恶性肿瘤的头号原因，在儿童及35岁以下成人中发病率位居第一[1]。

同时也是十大恶性肿瘤之一。

目前，白血病具体的发病原因至今尚未研究透彻，因此建立合适的白血病动物模型，对于白血病发病机制及药物研发具有重要意义。

本期为大家综述了白血病的基本情况及小鼠模型的分类、建立方法和应用。

第一章：白血病基本常识白血病是常见液体瘤白血病是常见的液体瘤，与结肠癌、肝癌等实体瘤不同的是，它是造血干细胞的异常分化和过度增殖导致，因此肿瘤细胞会遍布全身，会侵犯身体的每个脏器，造成全身衰竭。

造血干细胞是血液系统中的成体干细胞，具有长期自我更新和分化成各类成熟血细胞的能力。

如下图为造血干细胞可分类形成各种血细胞，如红细胞、血小板和白细胞：造血干细胞分化成各类血细胞（图片来自网站）白血病致病因素有哪些呢？现阶段认为白血病的发病因素：化学因素、电离辐射、药物、毒物、病毒、遗传因素等有关。

白血病主要分为四类根据白血病细胞的成熟程度和自然病程，白血病可分为急性和慢性两大类，临床上，白血病共分为四大类：急性髓系白血病（AML）、急性淋巴细胞白血病（ALL）、慢性髓系白血病（CML）和慢性淋巴细胞白血病（CLL）。

儿童白血病90%以上是急性的，其中急性白血病中70%～80%是ALL。

第二章：实验研究所用白血病模型首先，来了解一下常用的细胞株白血病中常用的小鼠品系用于建立白血病小鼠模型的小鼠可分为近交系和突变系。

根据不同类型和目的选择不同的小鼠品系，具体如下图所示：最后说说常用的动物模型，主要分为三类：一、异种移植模型异种移植模型是最常用的淋巴瘤动物模型。

根据实验目的选择相应的小鼠品系和细胞株后，通常细胞的接种方式为皮下注射、腹腔注射和尾静脉注射。

皮下注射和腹腔注射操作简单，很快在接种部位形成肿瘤或腹腔内形成多发性肿瘤，适合筛选针对白血病的药物。

脂多糖刺激巨噬细胞分泌含miR-155-5p_的外泌体促进肝星状细胞的活化及迁移

肝纤维化是肝炎-肝硬化-肝癌三部曲的重要病理表型，研究表明抑制肝纤维化能有效减缓肝炎向肝癌的进程［1］。

但目前临床上针对肝纤维化没有行之有效的治疗方案［2］。

因而，深入探索纤维化机制为寻找延缓乃至逆转纤维化的治疗靶点和节约医疗资源具有十分重要意义。

肝巨噬细胞在肝纤维化中发挥至关重要的作用，其主要功能与炎症、肝细胞损伤、肝星状细胞的活化和纤维化密切相关［3］。

肝星状细胞在肝脏生理和纤维生成起到关键作用，其能够受到肝巨噬细胞的调控［4］。

近来Lipopolysaccharide stimulates macrophages to secrete exosomes containing miR-155-5p to promote activation and migration of hepatic stellate cellsLIN Jiayi 1,LOU Anni 1,LI Xu 1,21Department of Emergency Medicine,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China;2Key Laboratory of First Aid and Trauma Research,Ministry of Education,Hainan Medical College,Haikou 571199,China摘要：目的探索脂多糖（LPS ）刺激下的巨噬细胞来源的外泌体对肝星状细胞的激活及迁移能力的影响及分子机制。

方法以100ng/mL 丙二醇甲醚醋酸（PMA ）处理人THP-1巨噬细胞24h ，诱导其分化为巨噬细胞，给予脂多糖刺激后收集巨噬细胞的培养上清，运用超速离心法提取外泌体并加以鉴定。

荧光定量PCR （qRT-PCR ）检测外泌体中miR-155-5p 的表达。

采用Transwell 共培养体系观察巨噬细胞分泌的外泌体对肝星状细胞LX2增殖、氧化应激、迁移和I 型胶原等纤维化标志物表达的影响。

肿瘤坏死因子偏高的原因

肿瘤坏死因子偏高的原因引言肿瘤坏死因子（TNF）是一种重要的细胞因子，它在炎症、免疫和肿瘤发生中起着重要作用。

然而，当肿瘤坏死因子的水平异常增高时，可能会引发一系列健康问题。

本文将探讨肿瘤坏死因子偏高的原因，并对其影响进行全面、详细、完整且深入地分析。

肿瘤坏死因子的作用肿瘤坏死因子是一种由多种细胞产生的细胞因子，包括巨噬细胞、T细胞和B细胞等。

它在炎症反应、免疫应答和肿瘤发生中发挥重要作用。

肿瘤坏死因子主要有两种形式：TNF-α和TNF-β。

它们通过结合细胞表面的受体，如TNFR1和TNFR2，来发挥作用。

TNF-α主要由巨噬细胞和T细胞产生，而TNF-β则主要由淋巴细胞产生。

肿瘤坏死因子在炎症反应中起着重要作用。

它能够引起血管内皮细胞的活化，导致炎症细胞的浸润。

此外，肿瘤坏死因子还能够诱导一系列炎症相关的细胞因子的产生，如白细胞介素1（IL-1）和白细胞介素6（IL-6），从而加剧炎症反应。

肿瘤坏死因子在免疫应答中也起着重要作用。

它能够促进巨噬细胞和其他免疫细胞的活化，增强它们对病原体的杀伤作用。

此外，肿瘤坏死因子还能够促进T细胞和B细胞的增殖和分化，从而增强免疫应答。

肿瘤坏死因子在肿瘤发生中也发挥重要作用。

它能够直接杀伤肿瘤细胞，并通过诱导细胞凋亡的方式抑制肿瘤生长。

此外，肿瘤坏死因子还能够诱导血管生成，从而促进肿瘤的生长和转移。

肿瘤坏死因子偏高的原因肿瘤坏死因子的水平异常增高可能是由多种原因引起的。

下面将对其中几个常见的原因进行详细探讨。

1. 炎症反应炎症反应是肿瘤坏死因子水平增高的常见原因之一。

当机体受到感染或损伤时，免疫系统会释放大量的肿瘤坏死因子来应对炎症刺激。

这种情况下，肿瘤坏死因子的产生增加，从而导致其水平升高。

2. 自身免疫性疾病自身免疫性疾病是指机体的免疫系统错误地攻击自身组织和器官。

在自身免疫性疾病中，免疫系统会释放过多的肿瘤坏死因子，导致其水平升高。

例如，类风湿性关节炎和系统性红斑狼疮等疾病都与肿瘤坏死因子的水平升高相关。

前列腺不典型小腺泡增生

2 A SA P与前列腺微小癌鉴别指标
ASAP与前列腺微小癌 (m inimal volume p rostatic adeno2 carcinoma,癌占活检组织总量的 5%以下 )之间的鉴别标准中 ,腺泡数目和病灶大小是最主要的一条 , ASAP腺泡的数目是癌腺泡数目的 2 /3 (11、17) , ASAP病灶比癌性病灶小一半 (014 mm、018 mm ) 。核增大、明显的核仁、核分裂象、腔内蓝色黏液及并存 P IN等形态特征在前列腺微小癌中更明显 ,但核深染及中～重度萎缩在 ASAP 比癌中更为常见 (分别为 44%、9%和 59%、35% ) 。 100%前列腺微小癌呈浸润性生长 ,但浸润性的生长方式也存在于 75%的 ASAP病例中。嗜酸性颗粒性分泌物与类晶体在两者无明显差异 [12 ] 。
前列腺癌占男性恶性肿瘤的第 2位 ,在发达国家 ,前列腺癌占全部恶性肿瘤的 19% ,在发展中国家为 513% [1 ] 。前列腺穿刺活检是发现和确诊前列腺癌的重要手段 ,但穿刺标本中经常会遇到少量不典型腺泡 ,疑似癌却又不能确定为癌 ,这便是前列腺不典型小腺泡增生 ( atyp ical small acinar p roliferation, ASAP) 。现将 ASAP形态特征、诊断标准、发病率、临床意义以及对发现前列腺癌的预测价值等作一综述。
1 A SA P的病理特征及应用现状
ASAP也称不典型腺体 ( atyp ia / atyp ical glands) [2 ] ,是由 Bostw ick等 [3 ]于 1993年首次提出的一个描述不典型腺样前列腺增生的诊断术语。4 年后这一诊断的临床意义得到首次阐述 [4 ] 。
ASAP为不典型腺泡病变 ,表现为排列紧密的灶性增生的小腺泡集落。这些小腺泡被覆一层几近透明的分泌细胞上皮 ,而基底细胞呈断片状或消失 (可经 34βE12 免疫组化证实 ) 。组织特点为 : ①有限数量的腺体 ; ② 极少腺体出现细胞不典型性 ,包括核增大、核仁增大 ; ③ 组织异型 :缺乏核异型的小腺泡杂乱无章地排列 ; ④ 腔内可见蓝色黏液、结晶体或粉红色蛋白样分泌物 [5 ] 。这些腺泡的结构形态和 /或细胞形态类似于分化较好的前列腺癌 ,但数量太少 ,只是怀疑为癌但不能明确诊断。不足以诊断为癌而做出 ASAP这一诊断主要见于两种情况 [6 ] : ①质的方面 ,缺乏足够的前列腺癌细胞和组织结构特点。例如一个病灶可能包括 12 个腺泡 ,腺泡缺乏基底细胞层 ,呈浸润性生长 ,但细胞形态和组织结构上尚未达到癌的诊断标准 (如缺少明显的核仁和明显的核增大 ) ; ②量的方面 ,包含的腺泡数量太少 ,腺泡的细胞和组织结构方面已经达到癌的诊断标准 ,但病灶的大小是其主要限制 (如 1～3个腺泡 ) 。

银杏达莫注射液联合骨髓间充质干细胞移植改善脑梗死后的神经功能

银杏达莫注射液联合骨髓间充质干细胞移植改善脑梗死后的神经功能杨朝阳【摘要】背景：通过细胞移植重建损伤脑组织成为治疗脑梗死的新途径，骨髓间充质干细胞成为近年来细胞移植治疗领域的研究热点。

目的：探讨银杏达莫注射液联合骨髓间充质干细胞移植对脑梗死大鼠神经功能的改善作用及相关机制。

方法：利用线栓法制作大鼠大脑中动脉闭塞模型，建模成功后60只SD大鼠随机分为对照组、细胞移植组及联合组。

对照组尾静脉注射PBS、细胞移植组尾静脉注射2.5×109 L-1的骨髓间充质干细胞悬液、联合组尾静脉注射2.5×109 L-1的骨髓间充质干细胞悬液和银杏达莫2 mL/kg，1次/d，连续注射5 d。

于移植后的1，3 d及1，2周进行mNSS行为学评分，以观察大鼠神经功能缺损状况。

移植后2周RT-PCR检测脑组织中脑源性神经生长因子、生长相关蛋白43基因表达变化，TUNEL法检测细胞凋亡情况，免疫组化法检测BrdU阳性细胞数。

结果与结论：移植后的1，3 d各组大鼠神经功能缺损评分差异无显著性意义(P >0.05)，在移植后1，2周，联合组神经功能缺损评分低于细胞移植组及对照组(P <0.05)；移植后2周，联合组脑源性神经生长因子、生长相关蛋白43 mRNA表达明显高于细胞移植组及对照组(P<0.05)，联合组凋亡细胞数目明显少于细胞移植组及对照组(P <0.05)，联合组BrdU阳性细胞数量明显多于细胞移植组及对照组(P <0.05)。

结果表明骨髓间充质干细胞联合银杏达莫干预能促进脑梗死组织脑源性神经生长因子、生长相关蛋白43 mRNA的表达，抑制细胞凋亡，改善大鼠神经功能。

%BACKGROUND:Reconstruction of damaged brain tissue through cel transplantation has become a new way to treat cerebral infarction. In recent years, bone marrow mesenchymal stem celshave become the new darling in cel transplantation therapy. OBJECTIVE:To investigate the effect of ginkgo-damole injection combined with bone marrow mesenchymal stem cel transplantation to improve the neurological function of acute cerebral infarction rats and its mechanism. METHODS:Animal models of middle cerebral artery occlusion were made in rats using suture method, and then 60 rat models were randomly divided into control group, cel transplantation group and combination group. The control group was given intravenous injection of PBSvia the tail vein; the cel transplantation group was given intravenous injection of bone marrow mesenchymal stem cel suspension (2.5×109/L) via the tail vein; the combination group was given intravenous injection of bone marrow mesenchymal stem cel suspension (2.5×109 /L) and ginkgo-damole injection (2 mL/kg, once a day, totaly 5 days)via the tail vein. Modified neurological severity scores were recorded at 1, 3 days and 1, 2 weeks after transplantation. At 2 weeks after transplantation, expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain were detected using RT-PCR; cel apoptosis detected using MTT assay; BrdU positive cels counted using immunohistochemistry method. RESULTS AND CONCLUSION:There were no differences in the modified neurologic severity scores among the three groups at 1, 3 days after transplantation (P > 0.05), but the modified neurological severity scores in the combination group were lower than those in the cel transplantation group and control group at 1, 2 weeks after transplantation (P < 0.05). The expressions of brain-derived neurotrophicfactor and growth associated protein 43 in the brain were significantly higher in the combination group than the other two groups at 2 weeks after transplantation (P < 0.05); compared with the other two groups, the number of apoptotic cels was less but the number of BrdU positive cels was higher in the combination group (P < 0.05). These findings indicate that the combination of ginkgo-damole injection and bone marrow mesenchymal stem cel transplantation can increase the expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain, inhibit cel apoptosis and improve neurological function in rats with cerebral infarction.【期刊名称】《中国组织工程研究》【年(卷),期】2015(000)050【总页数】6页(P8108-8113)【关键词】干细胞;移植;银杏达莫注射液;骨髓间充质干细胞;干细胞移植;脑源性神经生长因子;GAP-43;脑梗死【作者】杨朝阳【作者单位】济源市人民医院普内科，河南省济源市 454000【正文语种】中文【中图分类】R394.2文章亮点：1“干细胞循环”理论与中医理论中的“活血化瘀”法与有相通之处，银杏达莫注射液是从中药银杏叶中提取的复方制剂，能清除自由基、改善血液循环。

线粒体内参抗体：推荐Abbkine

线粒体内参抗体：推荐Abbkine COX IV抗体细胞⾊素c氧化酶(Cytochrome c oxidase，简称COX)是⼀种异源寡聚酶，通常含有13个亚基，定位于线粒体内膜，是线粒体呼吸链的酶复合物。

细胞⾊素c氧化酶中形成催化中⼼的3个最⼤的亚基由线粒体DNA编码，其余10个亚基包括COX IV由细胞核内的基因组DNA编码。

细胞⾊素c氧化酶(COX)活性缺陷和⼈类多种疾病相关。

COX IV抗体可⽤作线粒体蛋⽩上样量的内参抗体，其检测⽬标蛋⽩⼤⼩为16kD左右。

什么时候选择线粒体内参抗体？我们知道，在Western Blotting中使⽤内参其实就是在WB过程中的另外⽤内参对应的抗体检测内参蛋⽩，这样在检测⽬的产物的同时可以检测内参的表达，由于内参在各组织和细胞中的表达相对恒定，借助检测每个样品内参的量就可以⽤于校正上样误差，这样半定量的结果才更为可信。

此外使⽤内参可以作为空⽩对照，检测蛋⽩转膜情况是否完全、整个Western Blot显⾊或者发光体系是否正常。

常⽤的细胞总蛋⽩质内参有GAPDH和细胞⾻架蛋⽩β-Actin或β-Tubulin等。

对于⼀些植物的样本，则需要特别的植物Actin蛋⽩作为内参。

当实验样品中只是核蛋⽩，⽽不是细胞总蛋⽩提取液时，可以⽤组蛋⽩H（Histone H），或者增殖细胞核抗原（PCNA）等为核内参抗体。

⽽在⼀些特别的针对线粒体作为微环境的研究课题时，需要提取线粒体总蛋⽩并检测其中的⽬标蛋⽩含量，这时候常规的细胞总蛋⽩内参就不是很合适，我们推荐使⽤线粒体总蛋⽩的内参抗体，对于线粒体蛋⽩的检测，常⽤COX IV或VDAC1作为内参抗体，这⾥⾯COX IV⼜是被引⽤最多的线粒体内参。

COX IV的检测分⼦量⼤约在16kD左右，⽐较适合⽤于检测⽬标蛋⽩在较⼤的WB实验。

我们课题组是做凋亡和线粒体⽅⾯研究的，经常研究线粒体⽅⾯的通道蛋⽩，⼀直使⽤Abbkine的COX IV抗体来做WB，效价挺⾼的。

生物专业英语单词之欧阳学创编

Lesson One 细胞器的结构和功能Actin：肌动蛋白，是微丝的结构蛋白, 以两种形式存在, 即单体和多聚体。

basal body:：基体，真核细胞的纤毛或鞭毛基底部由微管及其相关蛋白质构成的短筒状结构，是纤毛和鞭毛的微管组织中心。

centriole：中心粒，动物、某些藻类和菌类细胞中的圆筒状细胞器，位于间期细胞核附近或有丝分裂细胞的纺锤体极区中心。

chemotaxis：趋化性，即由介质中化学物质的浓度差异形成的刺激所引起的趋向性。

chloroplast：叶绿体，绿色植物细胞内进行光合作用的结构，是一种质体。

chromosome：染色体，实质是脱氧核甘酸，为细胞核内由核蛋白组成、能用碱性染料染色、有结构的线状体，是遗传物质基因的载体。

cilia：纤毛，从一些原核细胞和真核细胞表面伸出的、能运动的突起。

cytoplasm：胞质，由细胞质基质、内膜系统、细胞骨架和包涵物组成。

cytoskeleton：细胞骨架，真核细胞中与保持细胞形态结构和细胞运动有关的纤维网络。

包括微管、微丝和中间丝。

dynein：动力蛋白，即纤毛中的一种具有ATP酶活性的巨大的蛋白质复合体。

endoplasmic reticulum：内质网，指细胞质中一系列囊腔和细管，彼此相通，形成一个隔离于细胞质基质的管道系统。

flagella：鞭毛，在某些细菌菌体上具有细长而弯曲的丝状物，是细菌的运动器官。

Golgi complex：高尔基复合体，由许多扁平的囊泡构成的以分泌为主要功能的细胞器。

lysosome：溶酶体，真核细胞中一种膜包围的异质的消化性细胞器。

是细胞内大分子降解的主要场所。

microfilament：微丝，由肌动蛋白分子螺旋状聚合成的纤丝，又称肌动蛋白丝，是细胞骨架的主要成分之一。

microtubule：微管，由微管蛋白原丝组成的不分支的中空管状结构，是细胞骨架成分，与细胞支持和运动有关。

mitochondrion：线粒体，真核细胞中由双层高度特化的单位膜围成的细胞器。

常用免疫学名词解释

附录：常用免疫学名词解释Aabsorption吸收应用特异性抗原与溶液中的抗体结合，形成不溶性复合物而除去抗体，例如用此法处理血清，即称为吸收血清。

用作吸收的抗原称为吸收剂。

accessory cell辅佐细胞特异性免疫应答需要的细胞，但不是实际介导的，通常用于描述抗原呈递细胞（APC）。

acquired immunity获得性免疫机体在生活过程中所获得的免疫力，称为获得性免疫，它与先天性免疫或天然免疫相反。

获得性免疫可分为：自动免疫，被动免疫，体液免疫与细胞免疫。

参看适应性免疫（adaptive immunity），过继性免疫（adoptive immunity），免疫耐受（immune tolerance）。

acquired immunodeficiency Sydrom（AIDS）获得性免疫缺陷综合征（艾滋病）由人类免疫缺陷病毒（HIV）所致的免疫缺陷病。

HIV感染主要引起T淋巴细胞CD4亚群的极度减少。

患者表现为迟发型超敏反应降低或消失，对机会感染菌极其易感，易发生某些少见的，如Kaposi氏肉瘤或Burkitt氏淋巴瘤。

HIV也可引起B 淋巴细胞多克隆性扩增，导致高丙种球蛋白血症。

尽管血清中免疫球蛋白量明显增加，但对抗原不能发生免疫反应。

这种综合征发生于“危险”人群，包括同性恋的男子，滥用静脉药物者，血液或血液制品的接受者，以及某些来自中非或加勒比海的人群。

在“危险”人群的异性伙伴中和AIDS母亲的婴儿中也已发现了这种综合症。

active immunization主动免疫（作用）抗原进入机体起免疫应答（自动免疫）adaptation tolerance适应性耐受生物在长期进化过程，宿主与寄生物在相互反应中，宿主的防御能力选择性地被减弱。

adaptative immunity适应免疫机体与抗原接触而发生的免疫力（包括主动体液免疫与主动细胞免疫）。

adherent cell粘附细胞在体外能粘附于表面的细胞。

关于细胞凋亡的讨论

关于细胞凋亡的讨论细胞调亡与坏死鉴别的简便方法midas1、PI和Hoechst33342双标：PI、Hoechst33342均可与细胞核DNA（或RNA）结合。

但是PI不能通过正常的细胞膜，Hoechst 则为膜通透性的荧光染料，故细胞在处于坏死或晚期调亡时细胞膜被破坏，这时可为PI着红色。

正常细胞和中早期调亡细胞均可被Hoechst着色，但是正常细胞核的Hoechst着色的形态呈圆形，淡兰色，内有较深的兰色颗粒；而调亡细胞的核由于浓集而呈亮兰色，或核呈分叶，碎片状，边集。

故PI着色为坏死细胞；亮兰色，或核呈分叶状，边集的Hoechst着色的为调亡细胞。

我最近在作的试验就是用的这种方法，附上一张我染的PC12细胞的图片，请大家指教。

更精确的定量可以通过流式细胞仪来检测。

用PI和Hoechst33342双标鉴别调亡、坏死，这种方法简便易行，结果比较可靠。

（图片见/bbs/post/view?bid=66&id=55585&tpg=1&ppg=1&sty=1&age=0#55585）2、PI和Calcein-Am双标：Calcein-AM 是一种绿色荧光标记物，可显示胞浆，为膜通透性的荧光染料。

Calcein-AM 一旦进入胞内，便可被内源性酯酶水解成绿色荧光物质calcein, 并保留在胞浆中。

细胞处于调亡时，由于核成碎片状，从而使此时的胞浆的calcein着色呈碎片状，但是PI为阴性。

细胞坏死时，胞浆的calcein染色基本上属于正常，但是PI为阳性，有时可以看到膜内有空泡。

但是晚期调亡细胞，胞浆有calcein着色并呈碎片状，而且PI为阳性。

3、PI和Annexin-V双标：Annexin-V（green）可以和胞膜内的磷脂酰丝氨酸（PS）特异性结合。

正常细胞膜的磷脂双分子层排列整齐，但是如果细胞损伤时，酯脂双分子层的排列就会被打乱，内层的可能会翻转到外层，Annexin-V就可以检测到这种现象。

巨噬细胞吞噬凋亡中性粒细胞后吞噬减少

Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic Neutrophils By Lars-Peter Erwig,Sharon Gordon,Garry M.Walsh,and Andrew J.ReesClearance of apoptotic neutrophils(polymorphonuclear leu-kocyte[PMN])by macrophages is thought to play a crucial role in resolution of acute inflammation.There is increasing evidence that ingestion of apoptotic cells modulates macro-phage behavior.We therefore performed experiments to determine whether ingestion of apoptotic PMN modulated the uptake process itself.Rat bone marrow-derived macro-phages(BMDM)ingested apoptotic PMN by a process that was enhanced by tumor necrosis factor(TNF)and attenu-ated by interferon(IFN)-␥,interleukin(IL)-4,and IL-10.It was inhibitable by the tetrapeptide arg-gly-gln-ser(RGDS),there-fore implicating the␣v␤3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with BMDM for30minutes,48 hours before rechallenge reduced uptake of apoptotic PMN by50%compared with previously unchallenged BMDM. Blocking initial uptake with RGDS abrogated the effect of parable and sustained attenuation of up-take was obtained by ligating␣v␤3with the monoclonal antibody(MoAb),F11,after a delay of more than90minutes, whereas MoAbs to CD25and CD45had no effect.Ligation of ␣6␤1and␣1␤2,integrins not previously implicated in the engulfment of apoptotic cells also decreased uptake with similar kinetics to F11.Therefore,apoptotic PMN regulate their own uptake through an integrin-dependent process, which can be reproduced by ligation of other integrins expressed by macrophages.௠1999by The American Society of Hematology.M ACROPHAGES INFLUENCE almost all aspects of immunological and inﬂammatory responses and play an essential role in linking the innate and acquired immunity.1 Macrophages not only induce injury,but also control key events in the resolution of inﬂammation and the repair processes that follow it.One of the critical functions in this process is phagocytosis of apoptotic cells via speciﬁc recognition mecha-nisms.To date,a number of recognition mechanisms for apoptotic cells have been described:(1)an uncharacterized lectin-dependent interaction2;(2)a complicated charge sensi-tive process involving the CD36/vitronectin receptor(␣v␤3) complex on the macrophage surface interacting with unknown moieties on the apoptotic polymorphonuclear leukocyte(PMN) surface via a thrombospondin bridge3,4;(3)a stereo-speciﬁc recognition of phosphatidylserine that is expressed on the surface of the apoptotic cell after loss of membrane asymme-try5,6;(4)macrophage scavenger receptors7;(5)the lipopolysac-charide(LPS)receptor CD148-10and macrosialin or CD68.11,12 The speciﬁc removal of apoptotic thymocytes,13eosino-phils,14and neutrophils15by macrophages has been well described.Extensive tissue damage and inﬂammation both precede and follow neutrophil death by necrosis.The cellular debris is phagocytosed by macrophages,which are activated by the process.In contrast,apoptosis of neutrophils is associated with the swift recognition of intact cells by macrophages followed by their ingestion and degradation.Local inﬂamma-tion and tissue injury are avoided not only because neutrophils are prevented from releasing their toxic contents,but also because the macrophages usual proinﬂammatory secretory response to phagocytosis is not activated16and may be biased towards release of the anti-inﬂammatory cytokine transforming growth factor(TGF)-␤.17These results suggest that uptake of apoptotic neutrophils by macrophages does not merely fail to induce synthesis of proinﬂammatory cytokines,but actively modulates macrophage function and biases the proﬁle of cytokines they release.This raises the question whether uptake of apoptotic cells‘‘imprints’’a pattern of behavior on macrophages analogous to the effect of exposure to some cytokines.18The speciﬁc purpose of the experiments described here was to ascertain whether uptake of apoptotic neutrophils modulates the ability of macrophages to ingest a second challenge with apoptotic PMNs.The results show a substantial reduction in the proportion of macrophages that ingest a second challenge of apoptotic PMNs,but that uptake can still be modulated appropriately by cytokines.The bone marrow-derived macrophages(BMDM)uptake of apop-totic PMN is RGDS-dependent and presumptively occurs by the ␣v␤3/CD36/thrombospondin pathway.After a delay of at least 90minutes,ligation of␣v␤3also downmodulates uptake of apoptotic cells speciﬁcally,and ligation of two other integrins,␣6␤1and␣1␤2,have the same effect.This shows that uptake of apoptotic cells is regulated by events that induce signalling through integrin receptors irrespective of whether or not they are directly associated with uptake.This raises the question whether uptake of apoptotic cells via␣v␤3reciprocally inﬂu-ences functions of integrins responsible for cell adhesion and facilitate the emigration of macrophages from an inﬂamed focus as described by Bellingan et al.19MATERIALS AND METHODSReagents.Recombinant human tumor necrosis factor(rhTNF)-␣, rhTGF-␤,and recombinant rat interferon(IFN)-␥were obtained from Boehringer(Ingelheim,Germany),Sigma Chemical Co(Dorset,UK), and Bradsure Biologicals Ltd(Loughborough,UK),respectively. Recombinant rat interleukin(IL)-4was produced in-house as described previously20using a Chinese hamster ovary(CHO)cell line generously donated by Dr Neil Barclay(MRC Cellular Immunology Unit,Oxford, UK).The rat monoclonal antibody(MoAb),F11,against the integrin␤3From the Department of Medicine and Therapeutics,University ofAberdeen,Aberdeen,UK.Submitted May8,1998;accepted October13,1998.Supported by Grant No.ER254/1-1from the Deutsche Forschungsge-meimschaft(to L.-P.E.),Grant No.044988/2/95/2from the WellcomeTrust(to G.M.W.),and the National Kidney Research Fund.Address reprint requests to Lars-Peter Erwig,MD,University ofAberdeen,Department of Medicine and Therapeutics,Institute ofMedical Sciences,Foresterhill,Aberdeen AB252ZD,UK;e-mail:L.P.Erwig@.The publication costs of this article were defrayed in part by pagecharge payment.This article must therefore be hereby marked‘‘adver-tisement’’in accordance with18U.S.C.section1734solely to indicatethis fact.௠1999by The American Society of Hematology.0006-4971/99/9304-0010$3.00/01406Blood,Vol93,No4(February15),1999:pp1406-1412chain21was a gift from Prof Michael Horton(Bone and Mineral Centre, University College London Medical School,London,UK).The mouse antirat integrin antibody␣6␤1,CD18,CD116,anti-CD45,anti-CD25, anti-ED3,and mouse antihuman CD21were obtained from Serotec (Oxford,UK).The rabbit antihuman erythrocyte membrane antibody was obtained from DAKO(Glostrup,Denmark).The tetrapeptides arg-gly-asp-ser(RGDS),arg-gly-glu-ser(RGES),and phospho-L-serine were obtained from Sigma Chemical Co.Isolation and culture of BMDM.Rat BMDM were obtained using a technique previously described in detail.22Brieﬂy,bone marrow cells wereﬂushed aseptically from the dissected femurs of male Spraque Dawley rats with a jet of complete medium directed through a25-gauge needle to form a single cell suspension.The cells were cultured in Dulbecco’s modiﬁed Eagle’s medium(DMEM)containing2mmol/L glutamine,100U/mL penicillin,and100U/mL streptomycin,10% heat-inactivated fetal calf serum,and10%L929conditioned medium as a source of macrophage-colony stimulating factor(M-CSF).After7 days in culture,BMDM were dispensed into24-well culture plates (Corning,Corning,NY)at a concentration of5ϫ105cells/well and rested in medium without added M-CSF for24hours before use in experiments.Inhibition of uptake of apoptotic neutrophils.BMDM were incu-bated with a series of inhibitors at concentrations of1mmol/L for15 minutes at4°C and were washed immediately before interaction with apoptotic neutrophils.Phospho-L-serine was used as a stereo-speciﬁc inhibitor of the macrophage phosphatidylserine receptor,using condi-tions described by Fadok et al.5The tetrapeptide arg-gly-asp-ser (RGDS)was used as described,4and the noninhibitory peptide arg-gly-glu-ser(RGES)added as a control.Assay for uptake of apoptotic neutrophils.BMDM were transferred to24-well plates at a density of5ϫ105cells/well and rested for24 hours before the medium was changed and the cells incubated with various cytokines.Uptake of apoptotic neutrophils was assessed after 48hours using a microscopically quantiﬁed phagocytic assay,which has previously been described and illustrated in detail.4,23Apoptotic neutrophils were prepared from PMN isolated from fresh heparinized normal human blood by dextran sedimentation and Percoll centrifuga-tion.They were aged in teﬂon bags for approximately24hours in RPMI 1640supplemented with antibiotics and10%fetal calf serum.More than98%of these cells excluded trypan blue while apoptosis was veriﬁed by oil immersion light microscopy of May-Giemsa–stained cytospin preparations as previously described.23The apoptotic cells were washed once and resuspended in RPMI at a concentration of2.5ϫ106/mL.A total of1mL of cells was added to each well and allowed to interact with the macrophages for30minutes at37°C in a5%CO2 atmosphere.The wells were washed in saline at4°C to remove noningested PMN,ﬁxed with2%gluteraldehyde in0.9%saline,and stained for myeloperoxidase to identify ingested PMN.The proportion of macrophages that had ingested neutrophils was then counted by inverted light microscopy.To determine the effect of previous ingestion of apoptotic neutrophils on macrophages,rat BMDM were transferred to24-well plates at a density of5ϫ105cells/well and rested for24hours before the medium was changed and cells were incubated with either medium alone,RGDS peptide followed by apoptotic neutrophils,or apoptotic neutrophils alone.After30minutes incubation,the wells were washed in saline at 4°C to remove noningested PMN,and the macrophages were rested for 48hours in control medium or medium containing various cytokines. They were then reincubated for30minutes with a second challenge of apoptotic neutrophils and the proportion of macrophages that took up PMN assessed.The assay for uptake of opsonized erythrocytes was performed exactly as previously described.24Ligation of the integrin receptors.MoAbs to␣v␤3,␣6␤1,␣1␤2, CD25,and CD45were used to assess the effect of ligation of macrophage cell surface receptors on uptake of apoptotic neutrophils.Macrophages were incubated at various MoAb concentrations ranging from0.01to10µg/mL,for30minutes,at4°C in saline,or were incubated with various concentrations of mouse antihuman CD21as an irrelevant isotype-matched control.The cells were then washed before incubation in medium for various times before the start of the standard interaction assay with apoptotic PMN.Quantitation of nitric oxide(NO)generation.Generation of NO was measured by assaying culture supernatants for nitrite,a stable reaction product of NO.Aliquots of200µL of each cell-free culture supernatant were incubated with50µL of Griess reagent(0.5% sulphanilamide,0.05%N-(1-naphtyl)ethylendiamine dihydrochloride in2.5%phosphoric acid)in96-ﬂat–bottomed tissue culture plates for 10minutes at room temperature.The optical densities of the assay samples were then measured at540nm using a solution of phenol red free DMEM.In most experiments,nitrite was measured48hours after exposure to cytokines.RESULTSCytokines regulate uptake of apoptotic neutrophils by BMDM. The initial experiment was designed to conﬁrm our previous observations that pro and antiinﬂammatory cytokines inﬂuence uptake of apoptotic human neutrophils by uncommitted rat BMDM.20TNF caused a36%increase in the proportion of BMDM that took up apoptotic neutrophils compared with controls,whereas IL-4,IL-10,and IFN-␥inhibited uptake by 56%,22%,and42%,respectively,and TGF-␤had no effect (Table1).Incubation with cytokines modulated not only the number of macrophages taking up apoptotic cells,but also the average number of neutrophils per macrophage,ie,IL-4caused a56%decrease in the number of macrophages taking up apoptotic neutrophils and a40%reduction in the number of neutrophils per macrophage.Theseﬁndings differ from those reported for human monocyte-derived macrophages.In these cells,incubation with proinﬂammatory cytokines(IFN and TNF)increased their ability to ingest neutrophils,whereas antiinﬂammatory cytokines(IL-4,IL-6,and IL-10)had no effect.25These differences could reﬂect the source and species of the macrophages used or the conditions in which they were matured.Recently,Bonder et al26have shown that human 7-day–cultured monocytes did not express the functionally active IL-2receptor␥-chain,a component of the IL-4receptor, whereas macrophages did,which may explain the different effect of IL-4on uptake of apoptotic neutrophils by monocyte-derived macrophages and BMDM.BMDM use an integrin-dependent mechanism to recognize apoptotic PMN.Human monocyte-like cell lines and murine peritoneal macrophages use the phosphatidylserine receptor Table1.Effect of Cytokines on the Number of Macrophages ThatTake up Apoptotic NeutrophilsCytokine(Concentration)Uptake of Apoptotic PMNs(%)Control31Ϯ2.8IFNϩTNF(20U/mL,10ng/mL)18Ϯ1.9*IFN(20U/mL)21.6Ϯ2†TNF(10ng/mL)42.6Ϯ2.8*TGF-␤(7.5ng/mL)28.2Ϯ2.5IL-4(5µL/mL)13Ϯ2.5*IL-10(100ng/mL)22.8Ϯ2.7Nϭ10.*PϽ.01relative to unstimulated controls.†PϽ.05relative to unstimulated controls.MODULATION OF NEUTROPHIL UPTAKE BY MACROPHAGES1407(PSR)for recognition of apoptotic cells.27Human monocyte-derived macrophages and murine BMDM have been reported to use the␣v␤3/CD36/thrombospondin pathway.3In our studies,1 mmol/L RDGS speciﬁcally inhibited uptake of apoptotic PMN by unstimulated rat BMDM and by macrophages incubated for 48hours with IFN-␥,TNF,IL-4,or TGF-␤.Neither the control peptide RGES,nor phospho-L-serine,which inhibits PS-mediated recognition of apoptotic cells,5had any effect on uptake by cytokine-stimulated or unstimulated macrophages (Table2).Thus,both uncommitted or cytokine-stimulated rat BMDM use an integrin-dependent recognition mechanism, presumptively the CD36/␣v␤3/thrombospondin system rather than a PSR-dependent mechanism.This conclusion is strength-ened by the demonstration of␣v␤3on the surface of BMDM by immunoﬂuorescence using the MoAb,F11,directed against the ␤3subunit of the receptor(data not shown).To verify the recognition mechanism,it would be necessary to block either CD36or␣v␤3on the macrophage surface.To our knowledge, the one antirat antibody available for this purpose is the mouse MoAb F11against the␤3subunit of the vitronectin receptor, which is a poor blocking antibody under our experimental conditions.BMDM incubated with F11for45minutes and then seeded in vitronectin-coated plates adhered as efficiently as control macrophages.There was no difference in the number ofnonadherent cells(less than1%of the seeded cells in both groups)when aliquots of the supernatants of control and F11-treated macrophages were examined2,4,12,and24hours after seeding(data not shown).Previous uptake of apoptotic PMNs reduces the ability of BMDM to ingest apoptotic PMN.To determine the effect of uptake of apoptotic neutrophils on macrophage function,uncom-mitted rat BMDM were challenged for30minutes with apoptotic neutrophils in medium alone or in the presence of RGD peptide to prevent uptake.They were then rested for48 hours in medium before being reexposed to freshly prepared apoptotic neutrophils.Macrophages that had previously in-gested apoptotic PMN had a markedly reduced ability to engulf apoptotic neutrophils compared with control macrophages (Fig1),whereas their ability to take up opsonized erythrocytes was unchanged(data not shown).The difference cannot be attributed to a nonspeciﬁc effect of the neutrophils because macrophages challenged with PMN in the presence of RGDS-peptide retain their subsequent ability to take up apoptotic neutrophils(Fig1).The degree of inhibition was comparable to that observed when BMDM are exposed to IFN-␥,IL-4,or IL-10,which we have previously shown cannot be reversed by treatment with TNF.28By contrast,uptake of apoptotic cells by BMDM did not abrogate the modulatory effects of TNF or other cytokines on uptake of apoptotic cells when added to the medium after the initial challenge.However,in each,their capacity to take up apoptotic PMNs was reduced by50%.Furthermore,prior uptake of PMNs did not affect the ability of IFN-␥to prime macrophages for generation of NO(Table3).In this set of experiments,there was no signiﬁcant difference in IFN/TNF-induced NO generation between uncommitted BMDM,macro-phages that had ingested apoptotic neutrophils,and macro-phages that have been incubated with RGDS peptide followed by apoptotic neutrophils.Thus,uptake of apoptotic cells speciﬁcally inhibits BMDM ability to engulf apoptotic cells without interfering with their ability to respond to a range of pro and antiinﬂammatory cytokines.Ligation of the␣v␤3receptor and other integrins reduce uptake of apoptotic PMNs.Overloading the macrophage phagocytic capacity provides the most obvious explanation as to why uptake of apoptotic cells prevented further uptake.Table2.Effect of Inhibitors on the Number of Macrophages Takingup Apoptotic PMN(%)ControlRGDS(1mmol/L)RGES(1mmol/L)Phospho-LSerine(1mmol/L)Control32Ϯ2.49.2*Ϯ130.6Ϯ228.2Ϯ2.2 IFN-TNF(20U/mL,10ng/mL)18.0Ϯ2.37.9*Ϯ1.517.9Ϯ4.816.3Ϯ3.2 TNF(10ng/mL)42.6Ϯ2.415*Ϯ1.839.7Ϯ3.143Ϯ3 TGF(7.5ng/mL)28Ϯ4.214.1†Ϯ2.127.5Ϯ3.324.8Ϯ3IL-4(5µL/mL)12.6Ϯ2.2 5.7†Ϯ1.913.5Ϯ112.1Ϯ1.3This table shows the effect of inhibitors on recognition of apoptotic PMNs by control and cytokine-stimulated BMDM.*PϽ.01relative to controls.†PϽ.05relative tocontrols.Fig1.Figure1shows the percentage uptake of apoptotic neutro-phils by BMDM.The macrophages were incubated48hours before the interaction assay with apoptotic neutrophils,RGDS followed by apoptotic neutrophils or medium.They were then washed and cultured in medium containing cytokines or medium alone before washing and a30-minute interaction with apoptotic PMN;mean؎standard error(SE),n؍10;*P F.01.Table3.Effect of Uptake of Apoptotic PMN or Ligation of Integrins on IFN/TNF-Induced NO Generation(Arbitrary Units)PMNRGDS(1mmol/L)ϩPMN MediumF11(1µg/mL)CD18(1µg/mL) IFN-TNF(20U/mL,10ng/mL)21.2Ϯ1.223.4Ϯ0.922.7Ϯ1.119.4Ϯ3.422.5Ϯ1.9 Control 2.1Ϯ1.2 2.9Ϯ0.5 2.5Ϯ0.7 2.09Ϯ0.9 1.8Ϯ1.11408ERWIG ET ALHowever,this seems unlikely for three reasons.First,uptake of opsonized erythrocytes did not downmodulate the ability of macrophages to ingest apoptotic neutrophils 48hours later (Table 4).Second,incubation of BMDM with neutrophils from different donors known to induce high or low uptake showed that irrespective of whether 20%or 40%of macrophages took up apoptotic cells and regardless of substantial differences in the number of PMN ingested per macrophage,the degree of downmodulation was about 50%(data not shown).Finally,the 48hours between the PMN challenges should be sufficient to allow the macrophages to recover.The fact that inhibition was prevented by incubation in the presence of RGDS suggested that the mechanism might involve the ␣v ␤3/CD36/thrombospon-din pathway.4This was addressed by incubation of BMDM for 30minutes with various concentrations of the MoAb,F11,against the ␤3subunit of the ␣v ␤3receptor.21F11blocks the calcium response after peptide binding to the vitronectin receptor in rat osteoclast.29It binds to ␣v ␤3on the surface of macrophages,but did not alter their adhesion to vitronectin,nor did it block uptake of apoptotic PMN by BMDM.Despite this,ligation of ␣v ␤3with F1112to 36hours before the interactionassay caused substantial reduction of PMN uptake (Fig 2),comparable to that induced by apoptotic PMN themselves.These data indicate that ligation of ␣v ␤3integrin decreases uptake of apoptotic neutrophils after a delay of at least 90minutes,whereas an isotype-matched control mouse antihuman CD21MoAb,which recognized neither PMNs nor macro-phages,had no effect.To examine the speciﬁcity of the effects of F11,the experiments were repeated,ﬁrst using MoAb against ␣6␤1,another integrin receptor expressed by macrophages,but not known to be involved in recognition of apoptotic neutro-phils,and secondly using a MoAb against CD45,another molecule on the macrophage plasma membrane.Strikingly the MoAb against ␣6␤1also downregulated uptake of PMNs with identical kinetics to antibodies against ␣v ␤3,whereas the MoAb against CD45had no effect (Fig 3).Thus,ligation of integrins,but not of other receptors on the surface of macrophages,speciﬁcally downregu-lates uptake of apoptotic cells after a delay of more than 90minutes,but did not affect the uptake of other particles such as opsonized red blood cells (data not shown).To conﬁrm these observations,we performed another set of experiments examining the role of receptor ligation on uptake of apoptotic PMNs by macrophages after ligation of the receptors for 45minutes 12hours before the interaction assay.Ligation of the integrin receptors CD11b (percentage uptake 14.4Ϯ2.1,P Ͻ.01)and CD18(13.4Ϯ3.4,P Ͻ.01)signiﬁcantly decreased uptake of apoptotic cells by macro-phages,whereas incubation with MoAbs against the IL-2receptor CD25(28.7Ϯ3.5)present on the macrophage surface,or ED3(29Ϯ4),only expressed by activated or tissueTable 4.Effect of Uptake of Opsonized Erythrocytes on the Number of Macrophages That Take up Apoptotic Neutrophils 48Hours LaterInitial ChallengeUptake of Apoptotic PMNs (%)Control25.8Ϯ7.3Opsonized erythrocytes 27.2Ϯ4.8N ϭ5.Fig 2.Figure 2shows the effect of ligation of ␣v ␤3(1␮g/mL)and an isotype-matched control CD21(10␮g/mL)on uptake of apoptotic neutrophils by BMDM.The macrophages were incubated for 30minutes at various times before the start of the interaction assay.Mean (percentage of uptake)؎SE,n ؍8.*P F.01.Fig 3.Figure 3shows the effect of ligation of ␣6␤1and CD45(10␮g/mL)on uptake of apoptotic neutrophils by BMDM.The macro-phages were incubated for 30minutes at various times before the start of the interaction assay.Mean (percentage of uptake)؎SE,n ؍8.*P F .01.MODULATION OF NEUTROPHIL UPTAKE BY MACROPHAGES 1409macrophages,were not different from controls(29.8Ϯ3.4). Thus,ligation of three different integrin receptors speciﬁcally downmodulated macrophage ingestion of apoptotic neutrophils, whereas ligation of two other receptors on the macrophage surface did not.It seems likely that at least some of the modulating effects of apoptotic PMNs themselves can be attributed to this mechanism.In addition,there was no signiﬁ-cant difference in IFN/TNF-induced NO generation between uncommitted BMDM and macrophages,which have been incubated with antibodies that ligate their integrin receptors (Table3).Thus,similar to uptake of apoptotic cells,ligation of integrin receptors speciﬁcally inhibits BMDM ability to engulf apoptotic cells without interfering with their ability to respond to a range of pro and antiinﬂammatory cytokines.DISCUSSIONThe speciﬁc uptake of apoptotic neutrophils by macrophages is one of the critical steps in the resolution of inﬂammation.30It provides a way to remove neutrophils before granulocyte lysis and release of the neutrophils’cytotoxic contents16and does not activate the macrophages usual proinﬂammatory response to phagocytosis.Indeed,Fadok et al17have recently provided evidence that uptake of apoptotic cells induces macrophages to synthesize the antiinﬂammatory cytokine,TGF-␤.The impor-tance of the process is illustrated by the observation that insufficient or impaired capacity for phagocytic clearance leads to disintegration of the cells undergoing apoptosis and worsen-ing of tissue damage.31We hypothesized that alterations in the process responsible for removal of apoptotic neutrophils might contribute to these observations.In some situations,induction of a single episode of acute inﬂammation resolves quickly, whereas a second episode results in progressive tissue damage. One explanation for this might be that the difference was caused by a reduced capacity to remove apoptotic neutrophils.32This prompted us to analyze the effect of ingestion of apoptotic cells on the ability of macrophages to take up a second pulse of apoptotic cells48hours later.The results show that uptake of the second pulse48hours after theﬁrst is consistently reduced by50%,which could have a substantial effect on tissue repair. The characteristics of the mechanisms responsible for im-paired uptake demonstrate that it involves speciﬁc interaction between the neutrophils and macrophages:(1)neutrophil up-take by BMDM was inhibited by RGDS,which interrupts integrin-dependent recognition;(2)it was not inﬂuenced by uptake of opsonized erythrocytes and was independent of the magnitude of theﬁrst‘‘neutrophil meal’’and thus unlikely to be due simply to macrophage‘‘indigestion’’;(3)the effect was sustained for at least48hours;(4)it did not interfere with cytokine-induced modulation of uptake of apoptotic cells;and (5)uptake of apoptotic neutrophils does not inﬂuence IFN-␥/ TNF-induced generation of NO by macrophages.Taken to-gether,these characteristics suggest that modulation is caused by the speciﬁc interactions between macrophage receptors and ligands on the PMN.A number of different macrophage receptor-mediated path-ways have been described to be involved in uptake of apoptotic neutrophils:(1)an uncharacterized lectin-dependent interac-tion2;(2)a complicated charge sensitive process involving the CD36/vitronectin receptor(␣v␤3)complex on the macrophage surface interacting with unknown moieties on the apoptotic PMN surface via a thrombospondin bridge3,4;(3)a stereo-speciﬁc recognition of phosphatidylserine that is expressed on the surface of the apoptotic cell after loss of membrane asymmetry5,6;(4)macrophage scavenger receptors7;(5)the LPS receptor,CD148-10and macrosialin or CD68.11,12Inhibition by RGDS,but not PS,suggests that uptake by rat BMDM in our experiments is mediated by the␣v␤3/CD36/thrombospondin recognition pathway,which has been extensively characterized by Savill et al.3,4The importance of␣v␤3was identiﬁed in blocking experiments using MoAbs.Our experiments were conducted using the MoAb,F11,an antibody to the␤3chain, which blocks some␣v␤3-dependent functions,but not the ability of BMDM to bind to vitronectin under our experimental conditions.Despite this,it caused a sustained downmodulation of the macrophages’ability to take up apoptotic cells after a delay of more than90minutes,comparable in degree to that seen after ingestion of apoptotic cells.This effect was not observed with isotype-matched control antibodies or with antibodies to CD25or CD45on the macrophage surface. Strikingly,however,antibodies to three other integrin receptors,␣6␤1,CD11b,and CD18,not known to be associated with uptake of apoptotic neutrophils,had the same effect.Thus, ligation of␤1,␤2,and␤3integrins all cause sustained speciﬁc downmodulation of uptake of apoptotic cells,but no effect on uptake of opsonized erythrocytes,and presumptively␤1and␤2 integrins downmodulate the function of␣v␤3in neutrophil uptake.There are precedents for cross-inhibition between integrin receptors,including interactions involving␣v␤3.Blystone et al33have previously shown that ligation of␣v␤3blocks high-affinity phagocytic function,but not adhesive function of theﬁbronectin receptor␣5␤1,possibly by inﬂuencing serine/ threonine kinase activity of the cytoplasmic portion␤1chain. Similarly␣4␤1ligation inhibits␣5␤1-dependent expression of metalloproteinases.34Diaz-Gonzalez et al35and Fenczik et al36 have analyzed the cross-talk between different integrins in detail and introduced the term transdominant inhibition to describe this phenomenon.They showed that ligation of␣ll b␤3 suppresses adhesive properties of␣5␤1and␣2␤1.35,36They demonstrated that the phenomenon was dependent on the assumption of the high-affinity state of␣ll b␤3and was attribut-able to conformational changes in the cytoplasmatic portion of the␤1chain.37It is not yet clear at what level changes in integrins might modulate uptake of apoptotic cells,partly because of uncertainties about the signalling pathways in-volved.The intracellular signalling pathways that control uptake of apoptotic cells have not been studied systematically.However, Rossi et al38have recently reported that activation of cyclic adenosine monophosphate(cAMP)signalling pathways by inﬂammatory mediators downmodulates macrophage ingestion of apoptotic cells and that alteration in cAMP concentrations might be responsible for the observation that ligation of CD44 speciﬁcally enhances phagocytosis of apoptotic neutrophils.39 Our results suggest that uptake is also regulated by cross-talk between integrins and emphasize the multiple levels of control for macrophage removal of apoptotic cells.The ability of macrophages to ingest apoptotic cells can be dynamically1410ERWIG ET AL。

谷胱甘肽提高梭菌活性

Engineering the robustness of Clostridium acetobutylicum by introducing glutathione biosynthetic capabilityLinjiang Zhu a,b,Hongjun Dong a,b,Yanping Zhang a,n,Yin Li a,na Institute of Microbiology,Chinese Academy of Sciences,No.1West Beichen Road,Chaoyang District,Beijing100101,Chinab Graduate School of the Chinese Academy of Sciences,Beijing100049,Chinaa r t i c l e i n f oArticle history:Received5October2010Received in revised form11January2011Accepted26January2011Available online4February2011Keywords:Clostridium acetobutylicumRobustnessGlutathioneAero-toleranceButanol toleranceButanol productiona b s t r a c tTo improve the aero-and solvent tolerance of the solvent-producing Clostridium acetobutylicum,glutathionebiosynthetic capability was introduced into C.acetobutylicum DSM1731by cloning and over-expressing thegshAB genes from Escherichia coli.Strain DSM1731(pITAB)produces glutathione,and shows a signiﬁcantlyimproved survival upon aeration and butanol challenge,as compared with the control.In addition,strainDSM1731(pITAB)exhibited an improved butanol tolerance and an increased butanol production capability,ascompared with the recombinant strains with only gshA or gshB gene.These results illustrated that introducingglutathione biosynthetic pathway,which is redundant for the metabolism of C.acetobutylicum,can increasethe robustness of the host to achieve a better solvent production.&2011Elsevier Inc.All rights reserved.1.IntroductionGlobal environmental problems and fuel crises have been promot-ing public attentions on the development of biotechnological processand alternative sources for the production of fuels and chemicals.Currently,a wide variety of chemicals,materials,and biofuels can beproduced by microorganisms from renewable biomass.This includesethanol,higher alcohols(Atsumi et al.,2008b;Zeng and Biebl,2002;Zhang et al.,2006),biodiesel(Kalscheuer et al.,2006),and alkanes(Schirmer et al.,2010).However,these metabolites are generally toxicto microbial cells,leading to a lowﬁnal fermentation titer(Nicolaouet al.,2010;Papoutsakis,2008).For example,wild type solventogenicClostridium acetobutylicum can rarely produce n-butanol higher than13g/L due to butanol toxicity(Jones and Woods,1986),while thebutanol produced by genetically engineered or chemically mutatedstrains of Clostridium rarely exceeded20g/L(Ezeji et al.,2010;Papoutsakis,2008;Sillers et al.,2008).n-Butanol can also be producedby engineered non-solventogenic bacteria,for instance by engineeredEscherichia coli(Atsumi et al.,2008a),Pseudomonas putida,and Bacillussubtilis(Nielsen et al.,2009),but theﬁnal titer of butanol has notexceeded1g/L.Moreover,several chemical stressants always co-existduring fermentation,resulting in overlapping stresses to cells(Nicolaou et al.,2010).Therefore,the robustness of C.acetobutylicumagainst multiple stresses needs to be improved.Robustness refers to the ability to maintain phenotypic stabilitywhen facing diverse perturbations(Kitano,2004).Microbial robust-ness coping with high osmolarity,salinity,and temperature can beimproved through overexpression of heat shock proteins,leading tosigniﬁcantly improved cryo-,thermo-,and solvent tolerance inLactobacillus(Desmond et al.,2004;Fiocco et al.,2007).Our previousstudies have shown that introducing glutathione(GSH)into Lacto-coccus lactis can improve the cellular resistance to oxidative,acid,andosmotic stresses(Li et al.,2003;Zhang et al.,2007;Zhang et al.,2010).Since GSH is also involved in protein stabilization,antioxidation,anddetoxiﬁcation(Lu,2009),we therefore wonder if introducing GSHbiosynthetic capability into C.acetobutylicum can improve its aero-tolerance and butanol tolerance,thus improving butanol production.To test this hypothesis,we engineered C.acetobutylicum for GSHproduction,and investigated the effect of the produced GSH oncellular physiology.2.Materials and methods2.1.Bacterial strains,plasmids,and culture conditionsThe bacterial strains and plasmids used are listed in Table1.E.coli strains were grown aerobically in Luria–Bertani(LB)Contents lists available at ScienceDirectjournal homepage:/locate/ymbenMetabolic Engineering1096-7176/$-see front matter&2011Elsevier Inc.All rights reserved.doi:10.1016/j.ymben.2011.01.009Abbreviations:g-GC,g-glutamyl-cysteine;g-GCS,g-glutamylcysteine synthe-tase;CFE,cell free extracts;CFU,colony forming unit;CGM,clostridium glucosemedium;Cys,cysteine;Cys-Gly,cysteinyl-glycine;GS,glutathione synthetase;GSH,glutathione;LB,Luria–Bertani;mBBr,monobromobimane;RBS,ribosomalbinding site;RCM,reinforced clostridial medium;SDS-PAGE,sulfate polyacryla-mide gel electrophoresisn Corresponding authors.Fax:+861064807485.E-mail addresses:zhangyp@(Y.Zhang),yli@(Y.Li).Metabolic Engineering13(2011)426–434medium supplemented with,when necessary,ampicillin(100m g/ mL)or chloramphenicol(35m g/mL). C.acetobutylicum strains were grown in an anaerobic chamber at371C.Liquid cultures were grown in reinforced clostridial medium(RCM)for routine growth,and mRCM medium(RCM containing20g/L glucose as the sole carbohydrate)for preparing competent cells(Dong et al., 2010).C.acetobutylicum strains harboring different plasmids were grown in RCM supplemented with erythromycin(25m g/mL).E.coli strains were stored atÀ801C in LB medium with10% glycerol.C.acetobutylicum strains were frozen atÀ801C in RCM with15%glycerol.2.2.DNA isolation,manipulation,and transformationPlasmids in E.coli were isolated using E.Z.N.A Plasmid Extrac-tion Kit,while genomic DNA of C.acetobutylicum and E.coli were isolated using E.Z.N.A Bacterial DNA Isolation Kit(Omega Biotek Inc.,Guangzhou,China).DNA restriction and cloning were per-formed according to standard procedures(Sambrook and Russell, 2001).Electrotransformation of C.acetobutylicum was carried out as previously described(Dong et al.,2010).2.3.Plasmids constructionPlasmid pITF that was derived from pIMP1with Pthl promoter and fdh gene(Dong et al.,2010)was used as a parent vector for cloning.E.coli JM109was used for vector construction.The gshA gene of E.coli(encoding g-glutamylcysteine synthetase,g-GCS) was ampliﬁed with the primers gA-F:50-TTCAGAGGATCCA-TCCCGGACGTATCACAGGCGC-30and gA-R:50-CTGCTGGCGCCT-CAGGCGTGTTTTTCCAGCCACAC(the bases underlined are the recognition site of restriction enzymes).The resulted1580-bp PCR product was cloned into the Bam HI and Bbe I sites of plasmid pITF to replace the fdh gene,yielding gshA expression plasmid pITA(Fig.1).The gshB gene(encoding glutathione synthetase,GS) was ampliﬁed from genomic DNA of E.coli with the primers gB-F:50-TTCAGAGGATCCATGATCAAGCTCGGCATCGTG-30and gB-R:50-AAGGCGAATTCTTACTGCTGCTGTAAACGTGCTTC-30.The 974-bp PCR product was cloned into the Bam HI and Eco RI sites of plasmid pITF to replace the fdh gene,yielding gshB expression plasmid pITB(Fig.1).The gshAB operon was created by fusion PCR using puriﬁed PCR products of gshA and gshB as templates.The primers used were gA-F,gAB-L:GCGGTGTGGCTGGAAAAACA-CGCCTAATTTAAGGAGGTTAAGAGGATGATCAAGCTCGGCATCGT-GATGG,and gB-R.The bases underlined in primer gAB-L are the ribosomal binding site(RBS)sequence.The2549-bp PCR product was cloned into the Bam HI and Bbe I sites of plasmid pITF to replace the fdh gene,yielding gshAB expression plasmid pITAB(Fig.1).Plasmids pITA,pITB,or pITAB were constructed in E.coli JM109,methylated in E.coli TOP10(pAN1),and then transformed into C.acetobutylicum DSM1731.Plasmid pIMP1 that does not contain any promoters was used as the empty vector control,and was also transformed into strain DSM1731.2.4.Preparation of cell free extracts and protein expression analysisTen milliliters fresh cultures were harvested by centrifugation (10,000g for10min at41C).Cell pellets were washed twice with pre-cooled saline(0.85%NaCl,w/v)and re-suspended in1mL of 200mM phosphate buffer(pH7.0)containing2mM EDTA.The cells were sonicated on ice for8min using a Soniﬁer S-450D (Branson Ultrasonics Corp.,Danbury,CT,USA)with the following protocol:4-s sonication with6-s interval,set at50%duty cycle. Cell debris was removed by centrifugation(12,000g for10min at 41C),resulting in cell free extracts(CFE).Total protein concentra-tions of CFE were determined using an RC DC protein assay kit (Bio-Rad)with bovine serum albumin as a standard(the standard error was less than10%).For protein expression analysis,CFE was mixed with an equal amount of2-fold concentrated loading buffer(10mmol/L Tris–HCl(pH6.8),4%(w/v)sodium dodecyl sulfate,20%(v/v)glycine,0.2%bromophenol blue,2% (v/v)2-mercaptoethanol).After boiling for10min,10m l of each sample was analyzed by sodium dodecyl sulfate polyacryla-mide gel electrophoresis(SDS-PAGE).The target bands in gel were excised and subjected to in-gel-digestion and MALDI–TOF MS analysis according to our previous report(Mao et al., 2010).2.5.Thiol assayThe monobromobimane(mBBr)ﬂuorescent labeling and HPLC methods were used to determine the intracellular and extracel-lular thiol(cysteine(Cys),g-glutamyl-cysteine(g-GC),and glu-tathione)concentrations according to the method described previously(Li et al.,2003).Ten millimolar stock solutions of GSH,g-GC,cysteinyl-glycine(Cys-Gly),and Cys in0.01mol/L HCl were prepared,aliquoted,and stored atÀ201C(to be used withinTable1Strains and plasmids used in this study.Strains and plasmids Relevant characteristics Reference orsourceStrainsC.acetobutylicum DSM1731Wild type,the parent strain in thisstudy,highly similar to C.acetobutylicum ATCC824accordingto our genome sequencing results (unpublished)DSMZC.acetobutylicum DSM1731(pIMP1)The plasmid control strain ofDSM1731,harboring pIMP1This studyC.acetobutylicum DSM1731(pITA)The gshA expressing recombinant,harboring pITAThis studyC.acetobutylicum DSM1731(pITB)The gshB expressing recombinant,harboring pITBThis studyC.acetobutylicum DSM1731(pITAB)The gshA and gshB expressingrecombinant,harboring pITAB,producing GSHThis studyE.coli JM109recA1mcrB+hsdR17Lab storageE.coliTOP10(pNA1)mcrA D(mrr–hsdRMS–mcrBC)recA1,applied for plasmid methylationbefore transformed into C.acetobutylicumInvitrogen,(Mermelsteinet al.,1992)PlasmidspAN1^3tI,p15a ori,Cm r Mermelstein et al.(1992)pIMP1The control plasmid without anypromoters,MLS r Amp r,shuttle vectorof E.coli-C.acetobutylicum Mermelstein et al. (1992)pITF MLS r Amp r,pIMP1derivative for fdhexpression under control of PthlDong et al.(2010)pITA pITF harboring gshA gene under Pthlpromoter,replaced the original fdhgene,MLS r,Amp rThis studypITB pITF harboring gshB gene under Pthlpromoter,replaced the original fdhgene,MLS r,Amp rThis studypITAB pITF harboring gshA and gshB geneunder Pthl promoter,replaced theoriginal fdh gene,MLS r,Amp rThis studyAbbreviations:Amp r,ampicillin resistance;Cm r,chloramphenicol resistance;MLS r,macrolide,lincosamide,and streptogramin B resistance;^3tI,^3TI methyltrans-ferase gene of Bacillus subtilis phage^3TI.Pthl,the promoter of thiolase gene inC.acetobutylicum;DSMZ,German Collection of Microorganisms and Cell Cultures,Braunschweig,Germany.L.Zhu et al./Metabolic Engineering13(2011)426–434427one month).Working standard solutions were freshly prepared from each stock solution by dilution in 0.01mol/L HCl.Fifty micromolar Cys-Gly was added to each sample as an internal standard.Five microliter mBBr-labeled samples were injected into a ZORBAX Eclipse XDB C18column (4.6mm Â250mm)packed with 5m m reversed-phase material (Agilent Technologies,Beijing,China)and with a C18guard column (4mm Â20mm,Agilent Technologies),using an Agilent Technologies HPLC 1200Series equipped a ﬂuorescence detector (Agilent Technologies,Beijing,China).The mBBr labeling procedure and HPLC analysis conditions were the same as described previously (Li et al.,2003).2.6.Butanol challenge experimentsFor butanol challenge experiments,RCM containing 19g/L butanol were prepared and ﬁlter-sterilized (0.2m m).Ten milliliter fresh culture (grown for 20h until early-stationary-phase)was centrifuged (5000g for 5min).Cell pellets were re-suspended in 10mL fresh RCM contained 19g/L butanol.The serially diluted cultures were plated onto the RCM agar (pH 5.8)and the colony forming unit (CFU)was determined after incubating anaerobically at 371C for 48h.Each experiment was carried out in triplicate.For butanol challenge growth experiment,RCM containing different concentrations of butanol (0–18g/L)were prepared and ﬁlter-sterilized (0.2m m).0.2mL fresh culture (OD 600¼1.0)of C.acetobutylicum DSM1731or its derivatives were inoculated into 10mL RCM containing different concentrations of butanol.The cultures were incubated anaerobically at 371C for 52h.Cell concentration was determined by measuring the turbidity at 600nm using a microtitre plate reader (SPECTRAmax PLUS 384;Molecular Devices,Sunnyvale,Calif.).Each experiment was car-ried out in triplicate.2.7.Aeration challenge experimentsTo determine if GSH can improve the aero-tolerance of C.acetobutylicum ,the effect of GSH addition in media on survival of DSM1731against aeration challenge was detected.Additionally,the aero-tolerance of strain DSM1731and its derivatives was compared.One milliliter fresh cultures (OD 600¼1.0)of C.acetobutylicum DSM1731and its derivatives harboring different plasmids were harvested by centrifugation (5000g for 5min)under anaerobic conditions.Cell pellets were washed with pre-cooled saline to remove the residual medium.Cells were centrifuged again and re-suspended in 10mL fresh RCM (adding different concentrations of GSH or antibiotics,when necessary)in 100mL ﬂasks.The culture was shaken at 200rpm,371C.Survival rate of C.acetobutylicum cells was calculated by dividing the CFU at a sampling time against the initial cell number.The serially diluted cultures wereplated onto the RCM Agar (pH 5.8);CFU was determined after incubating anaerobically at 371C for 48h.The CFU per mL for each sample were calculated.Each experiment was carried out in triplicate.2.8.Assay of fermentation phenotypeMetabolic phenotype of C.acetobutylicum was assayed in pH-controlled BioFlo 110bioreactors (New Brunswick Scientiﬁc,Edison,N.J.)with 4.0-L initial working volume as previously reported (Mao et al.,2010).Clostridium glucose medium (CGM)(Wiesenborn et al.,1988)was used as fermentation medium and supplemented with 25m g/mL of erythromycin and 0.15%anti-foam C (Sigma Chemical Co.,St.Louis,Mo.).The pH value (Z 5.0)was automatically controlled by the addition of 6M ammonium hydroxide.Nitrogen was introduced to maintain the anaerobic environments.The serially diluted cultures were plated on RCM plates with and without erythromycin to ensure the presence of the plasmid.Cell growth (OD 600)was determined with a UNICO UV/vis-2802PC Spectrophotometer (UNICO Instruments Co.,Ltd,Shanghai,China).The concentrations of acetate,butyrate,acetone,butanol,ethanol,and glucose were analyzed by an Agilent Technologies high-pressure liquid chromatography system 1200Series (Agilent Technologies,Beijing,China).A Bio-Rad (Bio-Rad Laboratories,Inc.,Hercules,Calif.)Aminex s HPX-87H ion exchange column (7.8mm Â300mm)with a Cation H guard column (4.6mm Â30mm)was used with a mobile phase of 0.05mM sulfuric acid ﬂowing at 0.50mL/min at 151C.A refrac-tive index (RI)detector (Agilent)was used for signal detection at 301C.The data displayed represent the mean value of two independent experiments.3.Results3.1.Addition of GSH improves the aero-tolerance of C.acetobutylicumAeration is quite severe for the survival of C.acetobutylicum .When DSM1731cells were incubated aerobically,cell growth immediately ceased.A 5log decrease in cell number was o bserved in the ﬁrst 3h of aeration,while no viable cells could be detected after 5h of aeration (Fig.2).Survival of C.acetobutylicum cells could be improved when GSH was added to the medium in the beginning of the culture (Fig.2).Exogenous addition of Cys also showed protective effect,but less effective as compared to GSH.Fig.2shows that addition of 3.2mM Cys is equivalent to addition of 0.8mM GSH,while addition of 19.2mM Cys is equivalent to the addition of 3.2mM GSH.This suggests that addition of GSH is about four to six times more effectiveasFig.1.Structure of plasmids pITA,pITB,and pITAB.Three recombinant plasmids were derived from the expression vector pITF (Dong et al.,2010),which contained Pthl promoter and an fdh gene.By replacing the fdh gene,target gene(s)(gshA ,gshB ,or gshA and gshB )were inserted under Pthl promoter,resulting in pITA,pITB,and pITAB,respectively.L.Zhu et al./Metabolic Engineering 13(2011)426–434428compared to the addition of Cys.We also determined the intracellular thiol concentration in C.acetobutylicum upon addi-tion of exogenous Cys or GSH (cultivated for 12h).When 19.2mM Cys or 19.2mM GSH was individually added to the medium,the intracellular Cys or GSH concentration reached 10.571.2nmol/mg and 5.670.7nmol/mg protein,respectively (GSH and Cys can be stably maintained in the medium and inside the cells at least for 28h,data no shown).On one hand,this shows that C.acetobutylicum is able to accumulate more Cys;on the other hand,it provides further evidence showing that GSH is more effective in C.acetobutylicum than Cys,as GSH functions much greater even in a lower intracellular concentration.3.2.Biosynthesis of GSH in C.acetobutylicum improves the aero-tolerance of the hostGSH is not present in most Gram-positive bacteria (Fahey et al.,1978)and is not detected in C.acetobutylicum DSM1731.The expression of target proteins of strain DSM1731and its derivatives were examined by SDS-PAGE.Fig.3A shows that g -GCS,GS,g -GCS,and GS were overexpressed in DSM1731(pITA),DSM1731(pITB),and DSM1731(pITAB),respectively.The relevant bands were excised and subjected to mass spectrometry identiﬁcation for validation.The band excised from the position of about 58kDa was identiﬁed to be a protein mixture that contains g -GCS from E.coli and ATP synthase F1(alpha subunit)from C.acetobutylicum according to peptide mass ﬁngerprint (Fig.3B).The band excised from the position of about 36kDa was identiﬁed to be GS from E.coli (Fig.3C).Introducing pITA into strain DSM1731resulted in the produc-tion of signiﬁcant amount of g -GC,and introducing pITAB resulted in the production of GSH and a minor amount of g -GC (Fig.4A).Introducing pITB into strain DSM1731did not change the thiol spectrum (Fig.4A),suggesting that C.acetobutylicum DSM1731lacks the activity of g -GCS,although a homolog encoding a putative g -GCS can be found in the C.acetobutylicum genome (CAC1539).Notably,although the expression level of GS in DSM1731(pITAB)(Fig.3A,lane 5)was much lower than that in DSM1731(pITB)(Fig.3A,lane 4),it did not affect the biosynthesis of GSH.Interestingly,strain DSM1731(pITAB)was able to secrete GSH to the medium (Fig.4B).When subjected to aeration,strain DSM1731(pITAB)exhibited signiﬁcantly improved survival rate,as compared with strain DSM1731harboring pIMP1,pITA,and pITB.Ten thousand fold difference in survival was observed between DSM1731(pITAB)and its empty vector control DSM1731(pIMP1)after 3h of aeration (Fig.5).Strain DSM1731(pITA)that was able to produce g -GC also showed an improved aero-tolerance,but to a less pronounced level as compared to strain DSM1731(pITAB)(Fig.5).It suggests that g -GC is also functional against oxidative stress,but less effective as compared toGSH.Fig.2.Survival of aeration-challenged C.acetobutylicum DSM1731when different concentration of GSH and Cys were added to the medium.One milliliter fresh culture (OD 600¼1.0)of C.acetobutylicum DSM1731was harvested,washed,and transferred to 10mL fresh RCM containing different concentrations of GSH and Cys,followed by incubating aerobically at 371C and 200rpm on a rotary shaker.CFU on the RCM agar plate after incubating anaerobically for 48h was counted to determine the survival.The GSH and Cys concentrations are shown in thegraph.Fig.3.Analysis of gene expression in strain DSM1731and its derivatives.(A)SDS-PAGE analysis of CFE of C.acetobutylicum DSM1731and its derivatives at OD 600of ne 1–5:wild type strain DSM1731and strain DSM1731harboring empty plasmid pIMP1,pITA,pITB and pITAB,respectively;Lane 0:protein marker.(B)The peptide mass ﬁngerprint of bands in the size of about 58kDa.The bands were identiﬁed as g -GCS from E.coli and ATP synthase F1(alpha subunit)from C.acetobutylicum .(C)The peptide mass ﬁngerprint of bands in the size of about 36kDa.The bands were identiﬁed as GS from E.coli .Mascot score greater than 53(the default MASCOT threshold for such searches)was accepted as signiﬁcant (p value o 0.05).L.Zhu et al./Metabolic Engineering 13(2011)426–4344293.3.Biosynthesis of GSH in C.acetobutylicum improves butanol tolerance and decreases growth inhibition by butanolTo test if GSH can protect C.acetobutylicum against butanol stress,cells of DSM1731(pIMP1/pITA/pITB/pITAB)were grown to early-stationary-phase,harvested,and exposed to 19g/L butanol challenge (Fig.6).No viable cells of strains DSM1731(pIMP1/pITA/pITB)could be detected after challenged with 19g/L butanol for 180min,while the viable cells of strain DSM1731(pITAB)remained 103CUF/mL.This indicates that GSH can protect the host against lethal butanol challenge.We also tested if GSH can help cells grow better in the presence of butanol.Cells of DSM1731wild type and DSM1731(pIMP1/pITA/pITB/pITAB)were grown in RCM containing different levels of butanol (up to 18g/L).No growth differences were found in RCM free of additional butanol (Fig.7A)or addition of 10g/L butanol (Fig.7B).As shown in Fig.7C and D,when challenged with high concentrations of butanol (14.5or 16.5g/L),strain DSM1731(pITAB)and its plasmid control strain DSM1731(pIMP1)obviously grew better than the wild type DSM1731and the single gene expression strains DSM1731(pITA)and DSM1731(pITB).Notably,strain DSM1731,DSM1731(pITA),and DSM1731(pITB)could hardly grow in the presence of 16.5g/L of butanol;while strain DSM1731(pITAB)could grow,and grew slightlybetter than DSM1731(pIMP1)(Fig.7D).At 18g/L of butanol challenge,none of the strains could grow (data not shown).The phenomenon that C.acetobutylicum strain harboring the empty vector pIMP1exhibited better growth ability under butanol stress has been reported,which was ascribed to the interaction between host and plasmid (Tomas et al.,2003;Walter et al.,1994).Strain DSM1731(pITAB)exhibited improved butanol resistance as compared to its control strain DSM1731(pITA)and DSM1731(pITB),suggesting that GSH synthesis can improve the butanol tolerance of the host.3.4.Biosynthesis of GSH in C.acetobutylicum improves butanol producing capabilityDuring pH-controlled batch fermentations,target plasmids could be stably maintained within the cell (data not shown).According to the proﬁles of cell growth and major metabolites (Fig.8),strain DSM1731(pITAB)produced 14.8g/L butanol,3.7g/LFig.4.The thiols concentration detected by HPLC.(A)Intracellular thiols concentra-tions of the ﬁve strains when they were grown to OD 600of 1.0in RCM medium.Cys:cysteine;g -GC:g -glutamylcysteine,GSH:glutathione.(B)Extracellular and intracellular GSH concentration proﬁles of strain DSM1731(pITAB)grown in RCM medium.GSH-o:extracellular GSH concentration;GSH-i:intracellular GSH concentra-tion (the unit of which was converted from nmol/mg protein to m mol/L by multiplying the cellconcentration).Fig.5.Survival of C.acetobutylicum DSM1731harboring different plasmids under aeration conditions.After anaerobically cultivated to an OD 600of 1.0,cells of different strains were individually transferred to fresh RCM,and incubated aerobically on a rotary shaker (200rpm)at 371C.Survival was determined after incubating anaerobically for 48h.Fig.6.Survival of C.acetobutylicum DSM1731harboring different plasmids under 19g/L butanol stress.After anaerobically cultivated to early-stationary-phase (20h),cells of different strains were individually transferred to fresh RCM contained 19g/L butanol,and incubated anaerobically at 371C.CFU was deter-mined after incubating anaerobically on RCM agar for 48h.L.Zhu et al./Metabolic Engineering 13(2011)426–434430acetone,and 1.2g/L ethanol (total solvents 19.7g/L with a ratio of 75:19:6)after 64h of fermentation (Table 2).The butanol pro-duced by strain DSM1731(pITAB)is 66%and 37%higher than its control strain DSM1731(pITB)and the wild-type strain DSM1731free of plasmid,respectively (Table 2).Although the individual solvent produced by strain DSM1731(pITAB)was only slightly higher than that of strain DSM1731(pIMP1)(total solvents 18.6g/L with a ratio of 72:19:9),solvent ratio was altered towards the direction of producing more butanol in the presence of GSH.The ﬁnal concentration of acetate and butyrate did not show signiﬁcant difference during the fermentation of the four strains.4.DiscussionGlutathione is the most prevalent non-protein thiol compound in living organisms that performs many diverse functions in metabolism (Lu,2009;Meister,1988)and stress resistance (Masip et al.,2006).However,most Gram-positive bacteria do not synthesize GSH,except for Streptococcus agalactiae ,Strepto-coccus pyrogens ,Enterococcus faecalis ,and Listeria monocytogenes (Gopal et al.,2005;Janowiak and Grifﬁth,2005;Sherrill and Fahey,1998).Some Gram-positive bacteria,including Leuconostoc kimchi and Leuconostoc mesenteroides ,alternatively accumulate g -GC rather than GSH (Kim et al.,2008).In E.coli ,GSH was synthesized through two ATP-dependent reactions,which were catalyzed by the enzymes of g -GCS and GS in turn.The E.coli g -GCS is a monomer of 58.3kDa (Huang et al.,1988)and GS is tetramer with four identical subunits if 35.6kDa (Yamaguchi et al.,1993),the size of which is almost equal to that of the expressed recombinant proteins shown in Fig.3A.Previously,we have introduced the GSH biosynthesis pathway into ctis subsp.cremoris NZ9000,resulted in an improved resistance to oxidative stress (Fu et al.,2006)and acid stress (Zhang et al.,2007)of the host.Although the protein encoded by CAC1539inthe genome of C.acetobutylicum is annotated as g -GCS;the fact that no g -GC was detected in C.acetobutylicum cells suggested this gene is not functional.No gene homologous to gshB was found in the genome of C.acetobutylicum ,this is consistent with the fact that introducing gshA into strain DSM1731only resulted in the production of g -GC but not GSH.Introducing gshAB into strain DSM1731resulted in the produc-tion of GSH,demonstrating for the ﬁrst time that,GSH can be produced by C.acetobutylicum and plays physiological roles.Although C.acetobutylicum is a typical obligatory anaerobe,it can grow under continuous microoxic conditions (5%O 2)(Kawasaki et al.,2004).In C.acetobutylicum ,only when dissolved oxygen accumulates so much that central metabolic enzymes like pyruvate–ferredoxin oxidoreductase (PFOR)are destructed,meta-bolism is halted and viability decreases (Hillmann et al.,2009a ).While GSH could protect some central metabolic enzymes like glyceraldehyde-3-phosphate dehydrogenase (GapA)in E.coli (Cotgreave et al.,2002;Leichert et al.,2008)and up-regulated synthesis of several glycolytic enzymes in ctis (Zhang et al.,2010),we postulate that GSH might also protect the central metabolism of C.acetobutylicum.C.acetobutylicum possesses several pathways for detoxiﬁcation of reactive oxygen species (ROS),including superoxide reductase (SOR)(Riebe et al.,2007),reverse rubrerythrin (revRbr)(Kawasaki et al.,2004),NADH rubredoxin oxidoreductase,and some glu-tathione peroxidase-like proteins (Kawasaki et al.,2005).Deletion of a peroxide repressor PerR-homologous protein resulted in a prolonged aero-tolerance,limited growth under aerobic condi-tions,and rapid consumption of oxygen from an aerobic environ-ment (Hillmann et al.,2008),demonstrating that PerR acts as a switch for aero-tolerance in C.acetobutylicum .Although naturally C.acetobutylicum does not synthesize GSH,the aero-tolerance of this strict anaerobe could be improved by adding external GSH or assembling a synthetic pathway for GSH biosynthesis.This shows that the aero-tolerance or even the ability to grow in thepresenceFig.7.Growth proﬁle of strain DSM1731and its derivatives under butanol-stressed conditions.After anaerobically cultivated to an OD 600of 1.0,cells of strain DSM1731and its derivatives were inoculated into fresh RCM containing different concentrations of butanol ((A)0g/L;(B)10g/L;(C)14.5g/L;(D)16.5g/L).Static cultures were performed anaerobically at 371C and cell concentrations were determined by measuring the turbidity at 600nm.Data represent the mean value of three independent experiments.L.Zhu et al./Metabolic Engineering 13(2011)426–434431。

洋参二醇皂苷对波动高糖诱导人脐静脉内皮细胞损伤的保护效应研究

论著洋参二醇皂苷对波动高糖诱导人脐静脉内皮细胞损伤的保护效应研究王景尚1，黄焊2，陈水龄2，殷惠军3(1.首都医科大学附属北京妇产医院，北京100026;2.中国中医科学院西苑医院，北京100091;3.甘肃中医药大学，甘肃兰州730000)[摘要]目的探讨洋参二醇皂苷对波动高糖诱导人脐静脉内皮细胞损伤的保护效应及其机制。

方法以体外培养人脐静脉内皮细胞为研究对象，通过每24 h间替更换高糖型0况1£肘全培养基（葡萄糖浓度25111111〇1/1〇与低糖型0：^1£：^1全培养基（葡萄糖浓度5.56111111〇1/L)构建人脐静脉内皮细胞血糖波动模型。

实验分为5组，包括正常低糖组、稳定高糖组、波动高糖组（间替高糖/低糖）、洋参二醇皂苷低剂量组（0. 04 mg/mL)、洋参二醇皂苷高剂量组(0. 08 mg/mL)。

细胞培养8 d，观察细胞形态学表现，MTT测定细胞成活率，取细胞培养上清液检测乳酸脱氢酶（LDH)、一氧化氮（NO)、内皮素-1(ET - 1)、可溶性细胞间黏附分子-1(sICAM - 1 )、肿瘤坏死因子-a (TNF - a)含量，细胞裂解液检测胞内超氧化物歧化酶（SOD) 活性和丙二醛（MDA)含量。

结果洋参二醇皂苷低、高剂量组细胞形态均较波动高糖组明显好转，细胞活性和细胞中SOD活性均明显高于波动高糖组（尸均<0.05)，LDH漏出率和细胞中T N F-a、sIC A M-l、E T-l、NO、MDA含量均明显低于波动高糖组（_P均<0.05)。

结论洋参二醇皂苷对波动高糖诱导的人脐静脉内皮细胞具有显著保护效应，其保护效应与其能够显著減轻葡萄糖波动诱导的氧化应激、炎症反应及细胞黏附分子的过度表达密切相关。

[关键词]洋参二醇皂苷；波动性高糖；人脐静脉内皮细胞；内皮损伤；保护效应doi：10.3969/j. issn. 1008 -8849.2017.05.001[中图分类号]R-33[文献标识码] A [文章编号]1008 - 8849(2017)05 -0457 -04 Study on the protection of Panax quinquefolium diol saponi on human umbilical vein endothelialcells injury induced by fluctuant high glucoseWANG Jingshang1 ,HUANG Ye2, CHEN Shuiling2 , YIN Huijun3(1. Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing 100026, China；2. Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091，China；3. Gansu University of Chinese Medicine, Lanzhou 730000, Gansu, China)Abstract：Objective It is to observe the protection of Panax quinquefolium diol saponi( DS) on human umbilical vein endothelial cells( HUYECs) suffering fluctuant high glucose and investigate the relative mechanism. Methods HUYECs were used as the research objects. Fluctuant high glucose was induced by a daily alternating 5. 56 or 25 mmol/L glucose. There were five groups ：normal glucose (NOR) , steady high glucose (SHG) , fluctuant high glucose (F H G)，low DS group (0.04 mg/mL,DSL) ,high DS group(0.08 mg/mL,DSH). HUYECs were incubated for 8 days. On the 9th day, the morphological changes were observed by light and electron microscopy, cell viability was measured by MTT method, cell membrane damage was determined by lactate dehydrogenase (LDH) leakage, the NO, ET-1, TNF-a, sICAM-1 concentrations in the cell culture supernatant were measured by ELISA method. The activities of SOD and the contents of MDA in the cell lysate were examined by enzyme method or spectrophotometry. Results Morphological results showed that both DSL and DSH cloud alleviate HUYECs injury induced by high blood glucose fluctuation obviously. The cell survival rates and cell viability were all significantly increased compared with FHG group ( F < 0. 05 ) . In comparison with FHG group, the levels of NO, MDA, ET-1, TNF-a, sICAM-1 in DSL and DSH groups were all significantly decreased(F <0. 05) , meanwhile the level of SOD was increased sig- nificantly(F <0. 05) .Conclusion Panax quinquefolium diol saponin(DS) can protect HUYECs suffering fluctuant high glucose obviously. The protective mechanism is closely related to its effects of relieving vessel oxidative stress, alleviating inflam- matory reaction and decreasing the expressions of cellular adhesion molecule.Key words：Panax quinquefolium diol saponin；fluctuant high glucose；human umbilical vein endothelial cells；endothe-lial injury;protection effect心血管并发症的发生是导致糖尿病患者致死、致残的首要病因，内皮功能异常是其发生发展的始动环节[1]。

腹水脱落细胞学检查的操作流程

腹水脱落细胞学检查的操作流程英文回答：Procedure for Cytological Examination of Ascitic Fluid.1. Collection: Ascitic fluid should be collected using aseptic technique, preferably by paracentesis. The sample should be placed in a sterile container and sent to the cytology laboratory as soon as possible.2. Preparation of Slides:Direct smear: A drop of ascitic fluid is placed on a glass slide and air-dried.Cytospin preparation: Ascitic fluid is centrifuged to concentrate the cells, which are then transferred to a slide using a cytospin centrifuge.3. Staining: The slides are stained using a Romanowsky-type stain, such as Wright-Giemsa or Papanicolaou stain.4. Microscopic Examination: The stained slides are examined under a microscope by a trained cytologist. The cytologist evaluates the cellular composition, morphology, and any abnormal findings.5. Interpretation: The cytological report typically includes a description of the cellular composition, any abnormal findings, and a diagnostic interpretation. The interpretation may include:Benign: No malignant or premalignant cells are identified.Malignant: Malignant cells are identified, indicating the presence of cancer.Suspicious: Atypical or inconclusive cells are present, requiring further evaluation.Non-diagnostic: The sample is inadequate or thecellular composition is not interpretable.中文回答：腹水脱落细胞学检查操作流程。

疟原虫

Abnormal hemoglobin and G6PD
deficiency groups protects against P.f.
Immunity
Acquired immunity
对再感染有一体液免疫和细胞免疫并存定的抵抗力；治疗后免疫力反复发作，免疫力上升逐渐消失． Premunition (带虫免疫) 免疫逃避
Prevention and cure
Treatment of infected individuals
Drugs: Chloroquine( 氯喹） ; pyrimethamine （乙胺嘧啶） ; anrtemisinine（青蒿素）etc Mosquito control
Protect susceptible population ： drugs: Vaccine:
Morphology
Ring stage
p.v. p.f.
Morphology
trophozoite
p.v.
Morphology
schizont
immature schizont mature schizont
p.v.
Morphology
Gametocyte p.v.
macrogametocytes (female) microgametocytes ( male)
红内期与临床发作有关
红内期M.P经数次裂体增殖后,部分裂殖子侵入RBC不分裂,直接发育成♀♂配子体
Life cycle
Brief summary of life cycle in human body
Mosquito stage
Sporozoites