这项工作是由美国国立卫生研究院资助P01 HL108801，R01-HL60234，R01-HL55330，R01-HL079904，以AMK财支持。 S. Ryter收到工资支持，从洛夫莱斯呼吸研究所。

注：该机构动物护理和使用委员会在Brigham和妇女医院/哈佛医学院区通过了以下程序。 1。盲肠结扎穿孔使用相同背景的小鼠（C57BL / 6），男，8〜10周龄。雌性小鼠比对败血症引起的致死的男性更耐。年长超过8周的小鼠产生比年轻小鼠在CLP后生存期少变的结果。约N = 10小鼠相比，每群应该用于存活分析。 N = 3-5老鼠是足够的自体吞噬分析。 <ol><li>用无菌小的手术器械（即手术刀，剪刀钝解剖钳），这应该是适用于啮齿动物手术。 </li><li>准备麻醉混合物：7.95毫升的PBS加150微升甲苯噻嗪（AnaSed注射100毫克/毫升）和700微升氯胺酮（Ketaset的&#160; CIII，100毫克/立方米升）。 </li><li>为了确保手术过程中无菌条件下，戴上手套，口罩和手术衣。 </li><li>称取动物。通过腹膜内注射麻醉他们（IP）在微升（10×体重（公克））的剂量提到的步骤3中的混合物中。例如，对于一个小鼠重22克，管理10x22 = 220微升的麻醉剂的混合物。因此，最终的剂量甲苯噻嗪和氯胺酮分别为17和80毫克/公斤体重。确保麻醉是否足够，没有屈曲下肢应脚趾捏后产生的注意 ：使用眼药膏对眼睛，防止干涩而在麻醉下。 </li><li>动物放置到一个干净的工作表面上他们的背上。尾巴和后爪是面向研究者。 ，使用醇（70％乙醇溶液） 注意消毒腹部皮肤：手术区域应完全剃光，以避免伤口污染通过与接触毛皮。此外，最佳地，一个循环热水垫应该被用来在外科手术过程中维持麻醉小鼠的正常体温。 </li><li>通过使用手术刀，做一个纵向切开皮肤平行的小1cm左右离开中线。不穿透尚未进入腹腔。用小剪刀将延长最初的切口。 </li><li>确定腹部肌肉和解剖他们能进入到腹膜腔。 </li><li>用钝解剖钳，找出盲肠并将其移出腹腔。要小心，不要损坏mesenterial血管（这可能导致致命的出血）。 </li><li>结扎的盲肠60％，实现了中档败血症。结扎等于或盲肠的75％以上通常导致高等级败血症，而结扎等于或盲肠结果在低档败血症小于25％。要小心，不要结扎回盲瓣（这可以阻止肠道连续性）。 </li><li>通过使用21号针头，由一个通和通穿刺（两孔）靠近结扎穿孔盲肠。穿孔的方向应该是从肠系膜至盲肠的抗肠系膜侧。要小心，不要刺破mesenterial血管。 </li><li>拔出针头。轻轻挤压盲肠结扎穿过两个孔，以确认通畅;只有少量的粪便应该通过它们被挤压。 </li><li>移动结扎和穿刺盲肠进入腹膜腔。 注：对于假老鼠，跳过步骤1.10-1.12。 </li><li>使用丝手术缝合线6-0与切割针，收腹部肌肉，使用丝手术缝合线6-0与切割针，收腹部皮肤。 </li><li>注入1毫升预热（37℃）生理盐水IP更换热和水化过程中丢失。将小鼠在热灯下，直到他们的复苏注意：理想情况下，一个更安全的加热装置（苏通道作为一个循环热水垫）可用于代替加热灯。 </li><li>术后注入buprenorphin（0.05毫克/千克体重SC），以达到镇痛注：动物应该有人看管，直到它恢复足够的意识，以保持胸骨斜卧。它经历了手术治疗的动物不应该被返回给其他动物的公司，直到完全康复。 </li><li>将动物放回笼子无限获得食物和水。当他们变得奄奄一息（由临床症状如未能触及时，移动或发绀表示）的小鼠将被安乐死。检查小鼠每隔4小时，以避免他们死之前安乐死。 </li></ol> 2。组织收获和固定<ol><li>制备4％多聚甲醛（PFA）解决方案：889毫升的PBS添加111毫升37％的煤灰。 </li><li>无论是使用二氧化碳诱导麻醉或氯胺酮/甲苯噻嗪的牺牲老鼠辨率在高剂量。 </li><li>固定小鼠到一个干净的工作台上。喷腹部和胸部的皮肤用70％乙醇溶液消毒了。 </li><li>使用解剖刀和小剪刀除去腹部和胸部的皮肤，以及皮肤覆盖气管区。 </li><li>通过去除其周围组织（肌肉和结缔组织）显示气管。 </li><li>将缝合线（不切割针）周围的气管。不要拧紧它。 </li><li>用一个小的外周静脉导管（20克），以“插管”气管。导管插入时要小心，避免气管撕裂。需要注意的是气管的最大直径是正下方的环状软骨。 </li><li>拧紧围绕气管/导管缝合。取下针头留在导管仅在塑料套管插入气管。只针担任导丝插入塑料套管。 </li><li>已抵押塑料插管插入气管后，切断日Ë腹部肌肉，打开腹部，并揭示了隔膜。 </li><li>使用针，穿刺膜片产生气胸（ 即插入空气进入胸膜腔）。肺部然后应该崩溃了。 </li><li>使用小剪刀，取出膜片，沿胸骨（胸骨）切割，取出肋骨，揭示了心脏和肺部。 </li><li>通过插入气管的塑料套管，固定有下30厘米H 2 O的压力的甲醛溶液的肺部。 </li><li>取出肺脏，把它们放入4％PFA 24小时，随后在70％的乙醇溶液中，直到处理。 </li><li>找出并消除心脏，肝脏和肾脏。以同样的方式，把它们放置到4％PFA中24小时，并随后在70％乙醇溶液，直至处理。 </li></ol> 3。 GFP小鼠和查看GFP的幻灯片<ol><li>如步骤1，术后过程中描述的GFP-LC3的小鼠进行电和假手术S作为在协议中的步骤2中描述。 </li><li>嵌入组织（即肺部，心脏，肝脏和肾脏）的石蜡和削减他们在连续5微米的部分。 </li><li>当准备好GFP染色，在60℃下30-60分钟，直到孵化石蜡熔化并组织出现半透明。 </li><li> Deparaffinize与标准渐变系列的二甲苯或二甲苯的代用品，5分钟二甲苯1，二甲苯2，二甲苯3然后3分钟的孵育在梯度系列的EtOH：100％，95％，75％，然后在蒸馏水中清洗。 注意：在脱蜡避免组织脱水，从而增加样品的背景。 </li><li>用柠檬酸缓冲液（10mM柠檬酸钠酸，0.05％吐温20，pH 6.0）中进行抗原修复，通过微波处理10-20分钟。请注意定期更换缓冲，以避免组织脱水。 </li><li>让溶液冷却在板凳上20分钟才能完成抗原修复。 </li><li>洗2〜3倍，用PBS。删除多余的缓冲和TRAnsfer从机架滑轨在滑框或其他加湿室，以防止干燥的水平位置。 </li><li>方框45-60分钟，在室温下用10％正常驴血清（也5％牛血清白蛋白，或来自在其中产生的二次抗体的动物优选血清）的PBS。 </li><li>孵育在初级GFP抗体样品以1:100稀释在PBS中过夜，在4℃下在潮湿的腔室中。轻轻地在组织中应用小片封口膜与抗体促进与组织均匀接触，防止蒸发。 注意：是在室温下进行余下的步骤。 </li><li>用PBS洗5倍，去除多余的一抗。孵化用在PBS中的第二抗体1:500-1:1,000为1-2小时，在室温下进行。 </li><li>用PBS洗5倍，去除多余的第二抗体。如果需要的话，染液的载玻片在此步骤。 </li><li>如果使用荧光二抗，孵育0.1％苏丹黑在70％乙醇f或15-20分钟，以减少自体荧光。擦去多余的苏丹黑周围组织，用PBS在20分钟内洗5倍。 </li><li>安装样品盖玻片和存储水平，直到安装解决方案集。 </li></ol> 4。替代方案：直接检测GFP-LC3的<ol><li>导管插入和膨胀的肺部有〜500毫升的第50/50 V / V十月PBS。 </li><li>通过把少量的OCT在一个cryomold的底部嵌入华侨城。 </li><li>小心地将解剖肺复杂的模具。 </li><li>慢慢地结冰甲基丁烷冷冻使用干冰。 </li><li>添加更多的华侨城，直到组织被完全覆盖，并冻结坚实。继续干冰储存在-80°C。 </li><li>用冷冻切片机，切5-10微米厚的部分保护部分的光。存储部分，并在-80℃下剩余的组织</li><li>为了准备幻灯片成像，从冰箱中取出部分，并立即申请下降的PBS，以防止干燥。 </li><li>固定30分钟，用4％PFA。用PBS冲洗。 </li><li>应用DAPI /赫司特10分钟。用PBS洗3次。 </li><li>安装样品盖玻片和存储水平，直到安装解决方案集。 </li><li>使用图像或落射荧光共聚焦显微镜。店内样品在4℃下长达一个月。 </li></ol> 5。组织电子显微镜的研制<ol><li>切组织为1mm左右的立方体固定。 </li><li>修复组织中的2％甲醛，2.5％戊二醛的0.1M二甲胂酸钠缓冲液，pH 7.4。 </li><li>用0.1M二甲胂酸钠缓冲液1-2小时洗。 注：固定组织可以在4℃下贮存于0.1M二甲胂酸钠</li><li>修复后在1％四氧化锇在1小时的0.2M二甲胂酸钠缓冲。 </li><li>在冲洗0.2M的二甲胂酸钠缓冲液10分钟3倍。 </li><li>脱水在70％乙醇，20分钟2倍。脱水以90％的乙醇，10分钟2倍。脱水在100％的乙醇，20分钟2倍。 </li><li> 10分钟孵育2个与环氧丙烷（环氧丙烷）。 </li><li> 1小时孵育与环氧丙烷/环氧树脂混合物（50:50）。环氧树脂混合物为：24克琼脂100树脂，13克十二烯基琥珀酸酐，13克甲基纳迪克酸酐和1ml的N-苄基二甲胺。使用木铲的一次性烧杯中调匀。 </li><li>孵育在未加帽的小瓶环氧树脂过夜（这允许任何残留的环氧丙烷蒸发）。 </li><li>嵌入标记胶囊与新配制的树脂。聚合在60℃下48小时。 </li><li>使用透射电子显微镜，在80或60千伏到电子显微镜照片薄膜的组织切片。打印的图像在相纸上或存储为数字媒体。 </li><li>分析自噬体形成核内含有完整细胞。低倍视野（五。克。2,000-4,000倍）可用于识别的有核细胞。通常6,800-10,000 X影像被用于第是自噬体的定量。计数单​​位面积自噬体15-30字段适当的统计表示。自噬体具有双层膜结构，其中晚期autophogosomes类似于填充空泡。 </li></ol>

<ol>
	<li>Hotchkiss, R. S., Karl, I. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathology+and+treatment+of+sepsis.">The pathology and treatment of sepsis.</a> N. Engl. J. Med. 348, 138-150 (2003).</li><li>Anaya, D. A., Nathens, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+severe+sepsis+in+secondary+peritonitis.">Risk factors for severe sepsis in secondary peritonitis.</a> Surg. Infect. 4, 355-362 (2003).</li><li>Bone, R. C., Grodzin, C. J., Balk, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis:+A+new+hypothesis+for+pathogenesis+of+the+disease+process.">Sepsis: A new hypothesis for pathogenesis of the disease process.</a> Chest. 112, 235-243 (1997).</li><li>Rivers, E., Med, s. h. o. c. k. .. N. .. E. n. g. l. .. J. .., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+goal-directed+therapy+in+the+treatment+of+severe+sepsis+and+septic+shock.">Early goal-directed therapy in the treatment of severe sepsis and septic shock.</a> N. Engl. J. Med.. 345, 1368-1377 (2001).</li><li>Deitch, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+intra-abdominal+infection.">Rodent models of intra-abdominal infection.</a> Shock. 24 (Suppl 1), 19-23 (2005).</li><li>Raven, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+sepsis+found+shockingly+lacking.">Rodent models of sepsis found shockingly lacking.</a> Nat. Med. 18, 998 (2012).</li><li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+pathogenesis+of+disease.">Autophagy in the pathogenesis of disease.</a> Cell. 132, 27-42 (2008).</li><li>Virgin, H. W., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+genes+in+immunity.">Autophagy genes in immunity.</a> Nat. Immunol. 10, 461-470 (2009).</li><li>Mizushima, N., Komatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+renovation+of+cells+and+tissues.">Autophagy: renovation of cells and tissues.</a> Cell. 147, 728-741 (2011).</li><li>Choi, A. M., Ryter, S. W., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+human+health+and+disease.">Autophagy in human health and disease.</a> N. Engl. J. Med. 368, 651-662 (2013).</li><li>Haspel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+macroautophagic+flux+in+vivo+using+a+leupeptin-based+assay.">Characterization of macroautophagic flux in vivo using a leupeptin-based assay.</a> Autophagy. 7, 629-642 (2011).</li><li>Chen, Z. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egr-1+regulates+autophagy+in+cigarette+smoke-induced+chronic+obstructive+pulmonary+disease.">Egr-1 regulates autophagy in cigarette smoke-induced chronic obstructive pulmonary disease.</a> PLos One. 3, 3313 (2008).</li><li>Kim, H. P., Chen, Z. H., Choi, A. M., Ryter, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+autophagy+in+clinical+tissues+of+lung+and+vascular+diseases.">Analyzing autophagy in clinical tissues of lung and vascular diseases.</a> Meth. Enzymol. 453, 197-216 (2009).</li><li>Flierl, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+functions+of+IL-17A+in+experimental+sepsis.">Adverse functions of IL-17A in experimental sepsis.</a> FASEB J. 22, 2198-2205 (2008).</li><li>Rittirsch, D., Huber-Lang, M. S., Flierl, M. A., Ward, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunodesign+of+experimental+sepsis+by+cecal+ligation+and+puncture.">Immunodesign of experimental sepsis by cecal ligation and puncture.</a> Nat. Protoc. 4, 31-36 (2009).</li><li>Mizushima, N., Yoshimori, T., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+mammalian+autophagy+research.">Methods in mammalian autophagy research.</a> Cell. 140, 313-326 (2010).</li><li>Wang, L., Ma, R., Flavell, R. A., Choi, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+mitogen-activated+protein+kinase+kinase+3+(MKK3)+for+activation+of+p38&#945;+and+p38&#948;+MAPK+isoforms+by+TGF-&#946;1+in+murine+mesangial+cells.">Requirement of mitogen-activated protein kinase kinase 3 (MKK3) for activation of p38&#945; and p38&#948; MAPK isoforms by TGF-&#946;1 in murine mesangial cells.</a> J. Biol. Chem. 277, 47257-47262 (2002).</li><li>Ding, Y., Kim, J. K., Kim, S. I., Na, H. J., Jun, S. Y., Lee, S. J., Choi, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-{beta}1+protects+against+mesangial+cell+apoptosis+via+induction+of+autophagy.">TGF-{beta}1 protects against mesangial cell apoptosis via induction of autophagy.</a> J Biol Chem. 285, 37909-37919 (2010).</li><li>Baker, C. C., Chaudry, I. H., Gaines, H. O., Baue, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+factors+affecting+mortality+rate+after+sepsis+in+a+murine+cecal+ligation+and+puncture+model.">Evaluation of factors affecting mortality rate after sepsis in a murine cecal ligation and puncture model.</a> Surgery. 94, 331-335 (1983).</li><li>Godshall, C. J., Scott, M. J., Peyton, J. C., Gardner, S. A., Cheadle, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+background+determines+susceptibility+during+murine+septic+peritonitis.">Genetic background determines susceptibility during murine septic peritonitis.</a> J. Surg. Res. 102, 45-49 (2002).</li><li>Carchman, E. H., Rao, J., Loughran, P. A., Rosengart, M. R., Zuckerbraun, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heme+oxygenase-1-mediated+autophagy+protects+against+hepatocyte+cell+death+and+hepatic+injury+from+infection/sepsis+in+mice.">Heme oxygenase-1-mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice.</a> Hepatology. 53, 2053-2062 (2011).</li><li>Lo, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lc3+over-expression+improves+survival+and+attenuates+lung+injury+through+increasing+autophagosomal+clearance+in+septic+mice.">Lc3 over-expression improves survival and attenuates lung injury through increasing autophagosomal clearance in septic mice.</a> Ann. Surg. 257, 352-363 (2013).</li></ol>

作者没有竞争经济利益申报

中电的主要优点是，它使研究人员能够（从低到中，高档IE）探讨不同严重程度的败血症。诱导的败血症的严重程度是受盲肠结扎的长度（这是最重要的决定因素），用于穿刺针的尺寸和孔的数量进行15。此外，小鼠品系和性别可以对脓毒症的严重程度影响;几株比其他人更容易与男性通常比女性19,20更容易。一起上述因素决定了电致死亡。 根据我们的经...

脓毒症，全身炎症反应感染，代表死亡的危重病患者1的首要原因。腹腔内感染，常常导致多种微生物脓毒症，占脓毒症病例的20％，其中有高达60％的2大量死亡。脓毒症相关的死亡率主要来自多器官功能障碍会导致随后的器官衰竭3,4。其他调查这种疾病的致病机理是目前急需推动新的和更有效的疗法的发展。 盲肠结扎穿孔（CLP）法是一种常用的方法， 在体内建模败血症。如盲肠是充分的细菌，其穿刺结果在多种微生物腹膜炎，易位细菌进入血液（菌血症），脓毒性休克，多器官功能障碍，并最终死亡5。人们普遍认为中电反映临床现实比以前的技 ​​术，例如注射内毒素或什至纯化的细菌进入啮齿动物更准确，因此，电被认为是金标准（尽管并非没有局限性）6对实验诱导，因此，脓毒症的发病机理的研究。在这本专着中，我们描述了旨在评估脓毒症的发病机制是否包括自噬协议。 吞噬，进化上保守的细胞的过程中，有利于受损的蛋白质和细胞器如线粒体的营业额，并播放在细胞内病原体包括细菌7,8的间隙中起重要作用。在自体吞噬，细胞质蛋白或细胞器螯合成双层膜结合的囊泡称为自噬体，其随后被输送到溶酶体降解9。一些蛋白质已被确定为自噬相关基因的哺乳动物同系物（署理）最初确定在酵母中，从而调节自噬过程。微管相关蛋白1轻链3B（LC3B）（的Atg8的同源物）从LC3B-I（自由格式）转换为LC3B-Ⅱ（磷脂酰乙醇胺结合的形式）表示在自噬体的形成9迈出的重要一步。自噬功能障碍与衰老和人类疾病包括癌症和神经退行性疾病相关的10。此外，自噬影响固有免疫和适应性免疫，如抗原提呈，淋巴细胞发育和细胞因子分泌免疫细胞8。因此，它似乎是合理的自噬作用也可能会在全身炎症反应感染（ 如败血症）发挥作用。 迄今为止的几种方法已经被描述，以评估自噬在组织损伤体内的作用。这些技术包括利用绿色荧光蛋白（GFP）-LC3表达的小鼠和自动的量化在组织的电子显微镜吞噬体（这两种方法都在本专着中描述）。另外的方法包括在组织匀浆自噬蛋白表达的定量，和自噬通量的分析（如在别处所述）11-13。本次审查的目的是评估在实验败血症的情况下自噬在体内提供当前协议。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >GFP-LC3 Transgenic Mice</td>
			<td >Riken (Japan)</td>
			<td >RBRC00806</td>
			<td >GFP-LC3#53</td>
		</tr>
		<tr >
			<td >Xylazine</td>
			<td >Henry-Schein</td>
			<td >568-0606</td>
			<td>Xylazine HCl Injection Vet</td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >Henry-Schein</td>
			<td >995-2949</td>
			<td>Ketaset Inj 100 mg/ml</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A405-20</td>
			<td>Histology Grade</td>
		</tr>
		<tr >
			<td >EtOH</td>
			<td >Fisher</td>
			<td>A407-1</td>
			<td>For Sterilizatiion</td>
		</tr>
		<tr >
			<td >silk surgical sutures 6-0</td>
			<td >Owens &#38; Minor</td>
			<td >2300-0078OG, 017624</td>
			<td></td>
		</tr>
		<tr >
			<td >buprenorphine-HCl</td>
			<td >Henry-Schein</td>
			<td >614-5157</td>
			<td>Buprenex Ampules</td>
		</tr>
		<tr >
			<td >paraformaldehyde (37%) solution</td>
			<td >JT Baker</td>
			<td >S898-09</td>
			<td></td>
		</tr>
		<tr >
			<td >xylenes</td>
			<td >Fisher</td>
			<td>X3P-1GAL </td>
			<td></td>
		</tr>
		<tr >
			<td >anti-GFP monoclonal antibody</td>
			<td>Life Technologies</td>
			<td>G10362</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoescht</td>
			<td >Sigma</td>
			<td>944403</td>
			<td></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306</td>
			<td></td>
		</tr>
		<tr >
			<td >OCT</td>
			<td >VWR scientific</td>
			<td >25608-930</td>
			<td></td>
		</tr>
		<tr >
			<td >Sudan Black</td>
			<td >Santa Cruz</td>
			<td >sc-203760</td>
			<td ></td>
		</tr>
		<tr >
			<td >EM grade Glutaraldehyde 2.5% in sodium cacodylate</td>
			<td >Electron microscopy Sciences</td>
			<td>15960</td>
			<td></td>
		</tr>
		<tr >
			<td >propylene oxide</td>
			<td >Sigma</td>
			<td>240397</td>
			<td></td>
		</tr>
		<tr >
			<td >Agar 100 resin</td>
			<td >Agar scientific</td>
			<td > R1045</td>
			<td></td>
		</tr>
		<tr >
			<td >dodecenylsuccinic anhydride</td>
			<td >Sigma</td>
			<td>46346</td>
			<td></td>
		</tr>
		<tr >
			<td >methylnadic anhydride</td>
			<td >Sigma</td>
			<td>45359</td>
			<td></td>
		</tr>
		<tr >
			<td >N-benzyldimethylamine</td>
			<td >Sigma</td>
			<td>185582</td>
			<td></td>
		</tr>
	</tbody>
</table>

cecal ligation and puncture-induced sepsis as a model to study autophagy in mice

败血症实验中可以使用的小鼠盲肠结扎穿孔（CLP）方法，这会导致多种微生物败血症诱发。在这里，一个协议提供了使用中电技术诱发不同程度的败血症小鼠。自噬是一个根本性的组织应激反应和病原体的入侵。二是目前的协议，以评估在实验败血症的情况下自噬在体内也这里。 （Ⅰ）表达绿色荧光蛋白（GFP）-LC3融合蛋白的转基因小鼠进行CLP。 GFP信号（泪点）的局部增强，因为无论是通过免疫组织化学或共聚焦分析测定，可用于检测增强的自噬体的形成，并因此改变了自噬途径的活化。 （II）的增强型自噬泡（自噬体）的每单位面积的组织的形成（如吞噬刺激的标记物）可以用电子显微镜进行量化。自噬反应败血症的研究是一个重要的排版onent理解由感染组织响应的机制。在这方面的研究成果可能对理解脓毒症的发病机制，这代表在危重病医学的一大难题，最终贡献。

菌血症存在于小鼠早中电诱发败血症14后6小时。败血症的临床症状（包括寒战，呼吸急促和运动障碍的活动）的程序后，出现约12小时。老鼠受到电开始诱导性腹膜炎后死在大约18小时。更严重的是脓毒症越增加的杀伤力15。具体而言，高档败血症导致在2-3天内死亡率为100％，而中档败血症导致大约60％的死亡率在CLP后7天（超出此时间限制，无死亡病例预计）（ 图1）。</strong...

Watch this Scientific Journal Video about Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice at JoVE.com

Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

实验性败血症可以在小鼠体内利用盲肠结扎穿孔（CLP）方法来诱导。当前的协议，以评估在CLP诱导的脓毒症的上下文中的自噬体内这里提出：一个协议，用于使用（GFP）-LC3的小鼠测定自噬和协议，用于测量自噬体形成通过电子显微镜。

盲肠结扎穿孔致脓毒症作为模型小鼠中自噬研究

Experimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is ...

cecal-ligation-puncture-induced-sepsis-as-model-to-study-autophagy

Research

JoVE Journal

Immunology and Infection

28.1K 次观看.  Brigham and Women's Hospital. 实验性败血症可以在小鼠体内利用盲肠结扎穿孔（CLP）方法来诱导。当前的协议，以评估在CLP诱导的脓毒症的上下文中的自噬体内这里提出：一个协议，用于使用（GFP）-LC3的小鼠测定自噬和协议，用于测量自噬体形成通过电子显微镜。 

视频: Cecal Ligation and Puncture-induced Sepsis as a Model To Study Autophagy in Mice

Colon Ascendens Stent Peritonitis (CASP) - a Standardized Model for Polymicrobial Abdominal Sepsis

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for establishing new therapeutic approaches in this field. Therefore, animal models are required that are able to closely mimic the human disease and also sufficiently deal with scientific questions. The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. In this model, a small stent is surgically inserted into the ascending colon of mice or rats leading to a continuous leakage of intestinal bacteria into the peritoneal cavity. The procedure results in peritonitis, systemic bacteraemia, organ infection by gut bacteria, and systemic but also local release of several pro- and anti-inflammatory cytokines. The lethality of CASP can be controlled by the diameter of the inserted stent. A variant of this model, the so-called CASP with intervention (CASPI), raises opportunity to remove the septic focus by a second operation according to common procedures in clinical practice. CASP is an easily learnable and highly reproducible model that closely mimics the clinical course of abdominal sepsis. It leads way to study on questions in several scientific fields e.g. immunology, infectiology, or surgery.

Colon Ascendens Stent Peritonitis CASP - a Standardized Model for Polymicrobial Abdominal Sepsis

The Colon Ascendens Stent Peritonitis (CASP) is a highly standardized model for polymicrobial abdominal sepsis in rodents. This article describes the surgical procedure of CASP. The CASP model and its variants allow the systematic investigation of various problems concerning the subject of sepsis.

科隆Ascendens支架腹膜炎（CASP） - 标准化的模式为多种微生物腹部败血症

Sepsis remains a persistent problem on intensive care units all over the world. Understanding the complex mechanisms of sepsis is the precondition for ...

科研

免疫与感染

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

解剖宿主 - 病毒相互作用模型疱疹病毒裂解性复制

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生儿多药脓毒症的对照小鼠模型

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

脓毒症诱导免疫抑制的切卡结扎和穿刺和鼻内感染双重模型的设计

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

利用无脊椎杆菌作为感染模型研究结核分枝杆菌复合物

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...