方法部分补充练习_22832.docx

MATERIALS AND METHODS (Role for Matrix Metalloproteinase 9 in Granuloma Formation during Pulmonary Mycobacterium tuberculosis Infection, from Cell, Vol. 148, Issue13, July. 2000)Mice. Specific-pathogen-free female, 6- to 8-week-old, CBA/J and C57BL/6 mice were purchased from Taconic (Germantown, NY), and wild-type FVB mice and MMP-9 null (MMP-9KO) breeder mice were purchased from Jackson Laboratory (Bar Harbor, ME). The MMP-9KO breeders were maintained as a colony at Colorado State University. All mice were maintained under barrier conditions with sterile mouse chow and water ad libitum. The specific-pathogen-free nature of the mouse colonies was demonstrated by testing sentinel animals, which were shown to be negative for 12 known mouse pathogens. All experimental procedures were approved by the Colorado State University Animal Care and Use Committee.小鼠 无特异病原体，6-8周大的 CBA/J 与 C57BL/6 雌性小鼠购自Taconic (Germantown, NY), 野生型FVB 小鼠与MMP-9 null (MMP-9KO) 育种小鼠breeder mice 购自Jackson实验室(Bar Harbor, ME)。MMP-9KO育种动物breeders 以群体形式as a colony 饲养于美国科罗拉多州大学。所有小鼠都隔栏under barrier conditions 喂养并随意喂无菌鼠食和水sterile mouse chow and water ad libitum。检测岗哨动物以证实小鼠群体的无特异病原体特征，结果表明12种已知的小鼠病原体均呈阴性。所有实验步骤均通过美国科罗拉多州大学动物保护使用委员会Colorado State University Animal Care and Use Committee同意.。 Infection with M. tuberculosis. Mycobacterium tuberculosis strain H37Rv, obtained from the Trudeau Mycobacterial Culture Collection, was passaged three times through pellicle and then stored frozen as a seed stock. Working stocks were made by passage through liquid culture of Proskauer-Beck medium containing 0.01% Tween 80, and aliquots of the organism in mid-log-phase growth were stored at 80C until needed. Mice were infected using procedures described previously (4). Briefly, bacterial stocks were diluted in 5 ml of sterile distilled water to 2 x 106 CFU/ml and placed in a nebulizer attached to an airborne infection system (Glass-Col, Terre Haute, IN). Mice were exposed to 40 min of aerosol, during which approximately 50 to 100 bacteria were deposited in the lungs of each animal. For the experiments in which mice were treated with BB-94 (also known as Batimastat), mice were infected with 500 CFU intratracheally (17). Bacterial load was determined by plating whole-organ homogenates onto nutrient 7H11 agar supplemented with oleic acid-albumin-dextrose-catalase. Colonies were enumerated after a 21-day incubation at 37C.M.肺结核感染 结核分支杆菌菌株H37Rv(购自Trudeau 细菌培养收集中心)通过菌膜进行三次传代后作为种源冰冻保存。用含0.01% 吐温80普-贝二氏培养基液体培养传代制备实验菌株,将中期培养生长的菌株各等分保存于80C条件下备用。小鼠感染模型程序如前所述(4)。简言之，菌群用5 ml无菌蒸馏水稀释至 2 x 106 CFU/ml后置于空气传播传染系统上的雾化器中 (Glass-Col, Terre Haute, IN)。使小鼠暴露于气雾剂环境40 min,每个小鼠肺中就有大约50至100 个细菌储存。实验中，小鼠用 BB-94 (也称之为巴马司他抗肿瘤药)处理,且支气管内感染500 CFU菌落单位(17)。细菌接种量通过在含有油酸-白蛋白-葡萄糖-过氧化氢酶的营养素7H11琼脂表面平皿培养完整器官组织匀浆而测定，并于37C中培养21天后进行菌落计数。Inhibition of MMP activity. BB-94 (Batimastat), a broad-spectrum inhibitor of MMP activity (17, 27) (see the supplemental material), was provided by British Biotech Pharmaceuticals Limited (Oxford, United Kingdom) and prepared for injection according to the manufacturers instructions. BB-94-treated mice were injected intraperitoneally with 0.2 ml of 20 mg of BB-94 per kg of body weight at the time of intratracheal infection with 500 CFU of M. tuberculosis and daily for the duration of the experiment.MMP活性抑制 BB-94 (巴马司他), 一种广谱MMP活性抑制剂 (17, 27) (见附加材料)由英国生物科技药物制剂有限公司(Oxford, United Kingdom) 提供，并根据生产厂家技术说明书制备成注射剂。在气管内感染500 CFU M.肺结核同时, BB-94-处理后的小鼠于实验期间每日腹膜内注射0.2 ml BB-94(按每公斤体重20 mg量计算)。Measurement of MMPs. The concentration of active MMP-9 was measured in lung homogenate supernatants using an MMP-9 Biotrak Activity Assay System (Biotrak Amersham Biosciences, Piscataway, NJ) according to the manufacturers instructions. Briefly, standards and samples were incubated in microtiter wells coated with anti-MMP-9 antibody. Any MMP-9 present remained bound to the precoated wells during subsequent washing and aspiration. MMP activity was detected through activation of the modified prodetection enzyme and the subsequent cleavage of its chromogenic peptide substrate. The resulting color was read at 405 nm on a spectrophotometer.MMPs测定 肺匀浆上清液中活性MMP-9的浓度测定应按照厂家提示使用MMP-9 Biotrak 活性测定系统(Biotrak Amersham Biosciences, Piscataway, NJ)完成。 简言之，标准品及样品在表面涂有抗-MMP-9抗体的微量滴定孔中培养。在冲洗和抽吸时要将出现的MMP-9保留于表面涂有抗-MMP-9抗体的滴定孔中。MMP 活性通过活化改良的prodetection酶及其产色肽底物分裂而测定，所得到的颜色用分光光度计于405 nm下测定。 Translate the following paragraph into English肺细胞消化 CO2窒息asphyxiation处死euthanize小鼠，然后打开肺腔。10 ml 含50 U/ml肝素heparin (Sigma-Aldrich, St. Louis, MO)的冷磷酸盐缓冲盐水cold phosphate-buffered saline灌注肺动脉将肺血冲洗干净。从胸腔取出肺组织，分开，用脱氧核糖核酸酶IV溶液 (Sigma-Aldrich; 30 g/ml) DNase IV (Sigma-Aldrich; 30 g/ml)和胶原酶D(Roche, Nutley, NJ; 0.7 mg/ml) （collagenase D (Roche, Nutley, NJ; 0.7 mg/ml) 在37C下处理30 分钟。肺部穿过细胞过滤器cell strainers制备单细胞悬浮 single-cell suspension(BD Biosciences, Mountain View, CA)。用Geys 溶液 (0.15 M NH4Cl and 10 mM KHCo3)溶解lyse残余的红血球，用含5%热灭活胎牛血清heat-inactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO)的RPMI (Invitrogen, Carlsbad, CA)冲洗细胞。用牛鲍氏计数法 (IMV International, Minneapolis, MN) Neubauer chamber (IMV International, Minneapolis, MN) 和 2% 锥虫蓝溶液 2% trypan blue solution测定活细胞viable cell总数。Lung cells digestion. Mice were euthanized by CO2 asphyxiation,and then pulmonary cavity were opened . Pulmonary artery was perfused by 10 ml cold phosphate-buffer saline containing 50 U/ml heparin(Sigma-Aldrich, St. Louis, MO) to wash out blood cells. Lung tissues were removed from chest, separated, and disposed by DNase IV (Sigma-Aldrich; 30 g/ml) and collagenase D(Roche, Nutley, NJ; 0.7 mg/ml) at 37 C for 30 minutes. Single-cell suspension(BD Biosciences, Mountain View, CA) was made by letting the lung tissues went through the cell strainers. The residual red blood cells were lysed with the use of Gey s solution (0.15 M NH4Cl and 10 mM KHCo3) and washed with the use of RPMI (Invitrogen, Carlsbad, CA)containing heat-inactivated fetal bovine serum (Atlas Biologicals, Fort Collins, CO).The viable cells amount was counted with the use of Neubauer chamber (IMV International, Minneapolis, MN) and 2% trypan blue solution.Flow cytometric analysis. Single-cell lung suspensions were stained with fluorescently labeled, monoclonal antibodies against CD3 and CD4 surface markers on T lymphocytes and CD11b, CD11c, and Gr-1 on macrophages. Antibodies were purchased from BD PharMingen (San Diego, CA) unless otherwise stated and were used at 25 g/ml. Cells were gated on lymphocytes or macrophages by forward and side scatter according to their characteristic scatter profile. Individual cell populations were identified according to the presence of specific fluorescence-labeled antibody, all analyses were performed with an acquisition of at least 30,000 events on a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using Cell Quest software (BD Biosciences, San Jose, CA).流式细胞检测分析 单细胞混悬液用荧光标记的T淋巴细胞表面的抗CD3、CD4表面标志及巨噬细胞表面的CD11b、CD11c和 Gr-1的单克隆抗体染色。除非特别指明，所有抗体都购自BD PharMingen (圣地亚哥, CA)，按25 g/ml使用根据特征散播图，细胞于淋巴细胞或巨噬细胞上通过向前和向侧边散播进行门控。单个细胞群根据特定的荧光标记抗体出现而鉴别。所有分析都基于Becton Dickinson FACSCalibur 流式细胞器 (BD Biosciences)至少30,000 次样本采集, 数据用Cell Quest 软件 (BD生物科学, San Jose, CA)分析。Cytometric bead array. A Cytometric Bead Array Mouse Inflammation kit (BD Biosciences, San Jose, CA) was used to measure TNF-, IFN-, and MCP-1 in the lung homogenate supernatants. The assay procedure was performed according to kit instructions, and the beads were analyzed on a FACSCalibur flow cytometer (BD Biosciences). 流式微球阵列用流式微球阵列小鼠炎症试剂盒(BD Biosciences, San Jose, CA)来检测肺(组织)匀浆上清液中TNF-、IFN-和MCP-1水平。分析程序根据试剂盒说明书，微珠采用FACSCalibur流式细胞器 (BD Biosciences)分析。Immunohistochemistry. Whole lungs from euthanized C57BL/6 and CBA/J mice were inflated with 10% formalin. The tissues were then paraffin embedded and sectioned. Following dewaxing, endogenous peroxidase was inhibited by incubation in 3% H2O2. Nonspecific staining was blocked with DAKO Protein Block Serum-Free (DakoCytomation, Carpinteria, CA). The cells containing cytokeratin were immunohistochemically stained with a polyclonal rabbit anti-cow cytokeratin antibody for wide-spectrum screening (DakoCytomation). A pretreatment of proteinase K (DakoCytomation) was utilized prior to antibody incubation. After incubation with primary antibody, the tissue sections were sequentially incubated with Dako Envision+ Rabbit System Labeled Polymer and horseradish peroxidase (DakoCytomation). Staining was developed with Liquid DAB+ (DakoCytomation), and cells were counterstained with hematoxylin. The antibody specifically stained epidermal keratin subunits of 58, 56, and 52 kDa; less abundant subunits of 60, 51, and 48 kDa were also found. For macrophage staining, anti-F4/80 antibody was used.免疫组织化学分析 从处死后的C57BL/6和CBA/J 小鼠 (n = 3)取出的全肺组织用10%福尔马林formalin膨胀inflated，然后用石蜡paraffin 包埋后切片embedded and sectioned。脱蜡dewaxing后，3%过氧化氢H2O2孵育抑制内源性过氧化物酶endogenous peroxidase 。DAKO Protein Block Serum-Free (DakoCytomation, Carpinteria, CA) 阻断非特异性染色 Nonspecific staining。含角蛋白cytokeratin细胞用多克隆兔抗牛角蛋白抗体polyclonal rabbit anti-cow cytokeratin antibody免疫组化染色后进行广谱扫描 (DakoCytomation)。抗体孵育前对蛋白酶K proteinase K (DakoCytomation)行预处理。经初级抗体孵育后，组织切片用Dako Envision和家兔系统标记聚合体辣根过氧化物酶(DakoCytomation) Rabbit System Labeled Polymer and horseradish peroxidase (DakoCytomation)进行连续孵化。细胞用DAB+ 液(DakoCytomation)染色，苏木精hematoxylin复染色counterstained 。发现表皮角蛋白亚单位epidermal keratin subunits 58、 56、52 kD，和相对少量的60、51、48 kDa亚单位经抗体特殊染色。用抗F4/80抗体进行巨噬细胞染色。Histological analysis. Whole lungs from euthanized FVB and MMP-9KO mice (n = 3) were inflated with 10% formalin and embedded in paraffin, and five 4-m serial sections were cut. Sections 1 and 5 were then stained with hematoxylin and eosin and examined by a veterinary pathologist with no prior knowledge of the experimental design.组织学分析 FVB和MMP-9KO 小鼠处死后(n = 3)取出其全肺组织用10%福尔马林膨胀并用石蜡包埋，连续5次切片，厚度为4-m。1号和5号切片经苏木精和伊红染色后送至对实验设计无先验知识的兽医病理学家进行组织学分析。 Statistical analysis. Statistical significance was determined with the two-tailed Student t test assuming unequal variances.统计分析 统计显著性用呈不等方差的双侧Student t检验验证。Translate the following passages into English 收集纯净培养(axenically cultivated ) HK-9株的滋养体(trophozoites)， 固定于4%多聚甲醛(paraformaldehyde)0.1M 缓冲液(pH 7.4)中。经离心(centrifugation) 冲洗, 1%温暖琼脂(agar) 加入沉淀物中。含滋养体固体琼脂(solidified agar)作1mm3切片(section) ， 并在各种底物(sediment) 中温育(incubate)， 以观察酶活性(enzyme activities)。然后标本以3%戊二醇(glutaraldehyde) 0.1M缓冲液(pH 7.4)后固定，用乙醇系列(ethanol series)脱水(dehydrate)，包埋(embed)于环氧树脂(epoxy resin)中。以超薄切片机(ultramicrotome)切片，用衍射电镜(transmission electron microscope)观察。切片用杞橼酸铝(lead citrate)染色。Trophozoites were collected from axenically cultivated HK - 9 strains, fixed in 4% paraformaldehyde 0.1 M buffer (pH 7.4),and washed by centrifugation. 1% Warm agar was put into the sediment. Solidified agar containing trophozoites was made into 1 mm3 section, which were incubated in a variety of sediment to observe enzyme activities .Then specimens were fixed in 3% glutaraldehyde 0.1 M buffer (pH 7.4), dehydrated by ethanol series, embed in epoxy resin, sliced with the use of ultramicrotome, observed by transmission electron microscope. Section were dyed by lead citrate.Rosiglitazone Evaluated for Cardiovascular Outcomes An Interim Analysis( from N Engl J Med,Vol 357 No 1 July 5 2007)METHODSPatientsThe RECORD study has been described in detail previously. We _ patients for the study from April 2001 through April 2003._ patients _ type 2 diabetes, as defined by criteria of the World Health Organization10; _ between the ages of 40 and 75 years; _ a body-mass index (the weight in kilograms divided by the square of the height in meters) of more than 25.0; and _ a glycated hemoglobin level _ _7.0% and _ or _ 9.0% while receiving maximum doses of metformin or a sulfonylurea. Exclusion criteria were the_ of other glucose-lowering (hypoglycemic) agents, _for a major cardiovascular event in the previous 3 months, a_, heart failure, _hepatic disease, renal impairment, and_ hypertension. The study protocol_ by ethics review committees or institutional review boards in accordance with the laws and customs of each country participating in the study._ was _from all patients.患者我们以前曾对RECORD 研究做过详细描述。我们从2001年4月至2003年4月为该研究招募患者。合格的病人患有2型糖尿病，符合世界卫生组织定义的标准，年龄在40-75岁，体质指数（体重的公斤数除以身高米数的平方） body-mass index (the weight in kilograms divided by the square of the height in meters) .，接受最大剂量二甲双胍或磺脲类药物metformin or a sulfonylurea治疗时的糖化血红蛋白水平glycated hemoglobin level .，但.。剔除标准为：当前使用其他降糖药，前个月内因重要心血管事件major cardiovascular event住院，计划接收心血管介入治疗 intervention，心衰，有临床意义的肝病，肾损伤，以及没有得到控制的高血压。伦理审查委员会或单位审查委员会ethics review committees or institutional review boards根据每个参加国的法律和惯例审核并批准了研究方案。全部患者都签署了知情同意书。Study DesignThe study is being conducted at 338 centers in 23 countries in Europe and Australasia. After a 4-week run-in period, patients who were already taking a sulfonylurea were randomly assigned to receive either additional rosiglitazone or metformin; those taking metformin were assigned to receive either additional rosiglitazone or a sulfonylurea (glyburide, gliclazide, or glimepiride, according to local practice). Random allocation was performed by telephone, with random permuted blocks stratified according to background medication.研究设计 本研究在欧洲和澳大拉西亚个国家的个中心进行。在周导入期后，已在服用磺脲类药物的患者被随机分配加用罗格列酮或二甲双胍；正在服用二甲双胍的患者被随机分配加用罗格列酮或一种磺脲类药物（根据当地实际情况使用格列苯脲、格列齐特或格列美脲）。根据患者的背景用药情况进行随机单位置换间距分层，然后通过电话进行随机分组。Throughout the study, the target glycated hemoglobin level was 7.0% or less. The starting dose of rosiglitazone (Avandia, GlaxoSmithKline) was 4 mg per day.The starting doses of metformin and sulfonylurea _according to _. If_ after 8 weeks of treatment, the doses of study drugs _to a _of 8 mg _ rosiglitazone, 2550 mg _ metformin, 15 mg _ glyburide, 240 mg _ gliclazide, and 4 mg _ glimepiride.If _ while patients were receiving the maximum tolerated dose, a third agent _ for patients in the rosiglitazone group or _ for patients in the control group. If patients _ triple therapy in the rosiglitazone group had _, the _ _that rosiglitazone be stopped and insulin therapy started.在整个研究期间，糖化血红蛋白目标水平为.。罗格列酮（文迪雅，葛兰素史克公司产品）rosiglitazone (Avandia, GlaxoSmithKline)的开始剂量为。二甲双胍和磺脲类药物 metformin and sulfonylurea的开始剂量根据当地惯常做法确定。如果治疗周后的糖化血红蛋白水平.，就将研究药物的剂量增至最大日剂量，罗格列酮为，二甲双胍metformin为，格列苯脲glyburide为，格列齐特 gliclazide为，格列美脲glimepiride为。如果患者接受最大耐受量maximum tolerated dose时的糖化血红蛋白水平.，则罗格列酮组患者加用第个药，对照组患者开始使用胰岛素。如果罗格列酮组接受药疗法triple therapy 患者的糖化血红蛋白水平.，则研究方案建议停用罗格列酮并开始胰岛素治疗。Outcome MeasuresThe primary end point was hospitalization (for acute myocardial infarction, congestive heart failure, stroke, unstable angina pectoris, transient ischemic attack, unplanned cardiovascular revascularization, amputation of extremities, or any other definite cardiovascular reason) or death from cardiovascular causes (including heart failure, acute myocardial infarction, sudden death, and death caused by acute vascular events including stroke); the outcome was analyzed as the time to first occurrence. Members of an independent committee evaluating clinical end points (five cardiologists, a neurologist, and a diabetologist) were unaware of study-group assignments and used prespecified criteria to adjudicate all potential outcomes reported by investigators. Evaluators in the trials contract organization (Quintiles) were unaware of study-group assignments in screening all serious adverse events for potential end points.转归指标主要终点是心血管原因造成的住院（因急性心肌梗死、充血性心力衰竭、卒中、不稳定性心绞痛、短暂脑缺血发作、计划外心血管血运重建术、截肢和其他任何明确的血管原因住院）或死亡（包括心衰、急性心肌梗死、猝死和包括卒中在内的急性血管时间导致的死亡）。根据至首次发生（事件）的时间进行转归分析。负责评估临床终点的独立委员会成员（5位心脏科医师、1位神经科医师和1为糖尿病科医师）不了解研究分组情况，采用事先制定的标准裁定研究者报告的全部可能性转归。该研究签约组织的评估者在不了解研究分组的情况下，筛查所有严重不良事件，确定可能的终点。 This interim report evaluated data that were available as of March 30, 2007. Secondary end points were death from cardiovascular causes and from any cause, myocardial infarction (resulting in either hospitalization or death), congestive heart failure (hospitalization or death), and the composite of death from cardiovascular causes, myocardial infarction, and stroke. Some events were pending adjudication while this report was being written. Analyses are reported both for adjudicated events only and for adjudicated events plus events pending adjudication. For 19 cardiovascular deaths pending adjudication, we cannot determine yet whether any were due to acute myocardial infarction or congestive heart failure.此中期报告评估了2007年3月30日以前获得的资料。次要终点是心血管原因和任何原因造成的死亡、心肌梗死（导致住院或死亡）、充血性心力衰竭（住院或死亡）以及由心血管原因造成的死亡、心肌梗死和卒中做成的复合终点。当我们撰写本报告时，某些事件正在等待裁定中。我们报告的分析中， 包括对已裁定事件的分析和对已裁定事件加待裁定事件的分析。对于有待裁定的19例心血管死亡，我们尚不能确定其死因是否为急性心肌梗死或充血性心力衰竭。Study OversightAn independent data and safety monitoring board meets twice annually to review unblinded safety data for the ongoing study; the most recent meeting took place on May 24, 2007. Members of the steering committee (seven academic investigators and one representative of the sponsor) developed the study design, had full access to the interim data, were responsible for the decision to publish the results, and wrote the manuscript. The committee members vouch for the accuracy and completeness of the data reported. Study committees and investigators are listed in the Appendix.研究监督独立的资料和安全监督委员会每年召开两次会议，审查仍在进行中的而研究的非盲法安全性资料。最近一次会议于2007年5月24日召开。指导委员会成员（7名学术研究者和1名资助方代表）制定研究设计方案，课自游查阅中期资料，负责决定结果的公布和撰写手稿。委员会成员保证所报告资料的准确性和完整性。研究委员会和研究者名单列于附录中。Statistical AnalysisThe RECORD study was designed as a noninferiori