在球蛋白细胞炎中进行半细胞外DNA半定量的受监督机器学习
Summary
细胞外DNA（ecDNA）在细胞死亡期间释放是消炎，并会导致炎症。在损伤部位测量ecDNA可以确定靶器官治疗的有效性。该协议描述了使用机器学习工具自动测量肾脏组织中 ecDNA。
Abstract
球状细胞死亡是骨髓过氧化物酶抗中性粒细胞质抗体相关血管炎（MPO-AAV）的病理特征。细胞外脱氧核糖核酸（ecDNA）在不同形式的细胞死亡期间释放，包括凋亡、坏死、坏死、中性粒细胞外陷阱（NETs）和肺气肿。测量这种细胞死亡是耗时与几个不同的生物标志物需要识别不同的生化形式的细胞死亡。ecDNA 的测量通常以血清和尿液作为肾损伤的代理进行，而不是发生在发生病理损伤的实际目标器官中。目前调查肾内肾肾脱氧核糖核酸的难点在于缺乏在实验和存档的人类肾脏活检中采用正式固定石蜡嵌组织（FFPE）的方法。该协议概述了在 FFPE 组织（人类和鼠）中染色 ecDNA 所需的步骤，使用公开提供的开源 ImageJ 插件可训练的 Weka 分段，在生成的图像中测量 ecDNA。可训练的Weka分段应用于球蛋白中的ecDNA，程序学习对ecDNA进行分类。此分类器应用于后续获得的肾脏图像，减少了对每个单个图像进行手动注释的需要。可训练韦卡分段的适应性在肾组织中进一步证明，从实验鼠抗MPO球状体炎（GN），以识别NET和ecMPO，抗MPO GN的常见病理贡献者。该方法对肾组织中的ecDNA进行了客观分析，明确证明了可训练的Weka分段程序能够区分正常肾组织和患病肾组织之间的疗效。该协议可以很容易地适应，以识别在其他器官的ecDNA，NET和ecMPO。
Introduction
骨髓环氧酶抗中性粒细胞质抗体相关血管炎（MPO-AAV）是一种自身免疫性疾病，导致肾功能衰竭的病理球状损伤与相当大的细胞死亡和脱氧核糖核酸（DNA）释放11，2。2DNA可以作为危险信号激活免疫系统。在正常健康条件下，DNA的核位置可以保护他们免受免疫系统的照射。自我DNA在致病过程或自身免疫过程中在细胞外释放，被免疫系统视为一种有效的亲炎损伤相关分子自蛋白（DAMP）3。额外的细胞DNA（ecDNA）通过几个不同的机制从垂死细胞中释放出来，这些机制受不同的生化途径控制，如凋亡、神经球菌外细胞陷阱形成（NET）、坏死或肺尘埃增多4,4、5、6、7、8。5,6,7,8
我们在此描述从死亡细胞释放的ecDNA的方法，从实验抗MPO GN和从MPO-AAV99，1010患者患者体内固定石蜡嵌入（FFPE）肾脏部分释放。从血清和尿液以及体外测定11，12,12检测中检测循环双链DNA（dsDNA）和DNA复合物的方法有多种。这些方法虽然准确确定ecDNA的含量，但不能确定在解剖学上释放ecDNA的位置。有一些方法描述了ecDNA的具体测量，如凋亡调谐器和细胞碎片的测量13，14。13,没有任何方法描述测量ecDNA在发生病理损伤的FFPE肾脏中所有形式的细胞死亡。这一点很重要，以确定实验性治疗是否正在清除从实际目标器官的病理损伤部位的ecDNA。
从肾脏样本中采集多个图像可创建大量数据，通常由一个用户进行分析。这耗费人力，耗时，由于用户偏见，其他用户可能会难以重现。可训练Weka分段是ImageJ的开源软件插件，它使用尖端的生物信息工具使用机器学习算法15、16,16对像素进行分类。此方法是”可训练”的，它根据用户对像素段的分类来学习，并将新学习的分类应用于其他图像。此方法依赖于 ImageJ 程序中的常见分析工具，这些工具用于将段中的每个像素”分类”为属于特定”类”。程序学习”分类器”后，可用于识别同一图像中的其他类似分类段。然后，此模型将保存并应用于同一实验中的其他图像集。
目前确定肾分部原位ecDNA的障碍是内源性自荧光，从正式固定在形式和劳动密集型分析的图像。我们在这里描述如何淬火这种自动荧光，检测ecDNA，并使用监督机器学习进行高通量测量ecDNA。我们之前已经发布了使用 ImageJ 中的宏进行的 NET 和细胞外 MPO （ecMPO） 的测量，现在我们演示了这些方法使用监督机器学习1的半自动化。我们演示了机器学习工具的适应性，以在同一图像中对 NET 和 ecMPO 的替代污渍进行分类。此处描述的这些染色方法用于检测 ecDNA、NET 和 ecMPO，可转换为其他固体器官和疾病，其中 ecDNA、NETS 和 ecMPO 在使类风湿性关节炎和狼疮等,疾病长期存在中发挥作用。
Protocol
Representative Results
Discussion
存在多种方案，用于测量肾上腺炎患者和小鼠模型血清和尿液中的消炎标记物。这种所述协议允许直接分析球状体内的细胞死亡产物（ecDNA、NET和ecMPO）。该协议中最重要的步骤是组织准备和成像。使用荧光染色方法进行分析的主要限制因素是组织自荧光。形式内固定石蜡组织受到自荧光，可以掩盖特定的荧光染色。染色方法的最后一步，其中幻灯片浸入苏丹黑色，衰减组织的自荧光，并允许通过…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们承认莫纳什微成像用于使用尼康C1直立共聚焦激光扫描显微镜和莫纳什组织学平台处理肾脏组织。
Materials
| Bovine Serum Albumin | SIGMA | A2153 | 5% and 1% solutions are made up in PBS, can be made in bulk and frozen- discard once thawed. |
| Chicken anti Goat IgG (H+L) cross absorbed antibody Alexa Fluor 594 | ThermoFisher Scientific | A-21468 | Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail |
| Chicken anti mouse IgG (H+L) cross absorbed antibody, Alex Fluor 647 | ThermoFisher Scientific | A-121468 | Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail |
| Chicken anti rabbit IgG (H+L) Cross absorbed antibody Alexa Fluor 488 | ThermoFisher Scientific | A-21441 | Spin in mini centrifuge for 1 minute prior to use to avoid any free conjugate in your antibody cocktail |
| Chicken sera | SIGMA | C5405 | Made up in 1%BSA/PBS |
| Coverslips 24 x60 mm | Azerscientific | ES0107222 | #1.5 This is not standard thickness- designed for use in confocal microscopy |
| EDTA 10mM | SIGMA | E6758 | Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x Solution |
| Ethanol 30%, 70% and 100% | Chem Supply | UN1170 | Supplied as 100% undenatured ethanol- dilute to 30% and 70% using distilled water |
| Formaldehyde, 4% (10% Neutral buffered Formalin) | TRAJAN | NBF-500 | Kidney is put into a 5ml tube containing 3ml of formalin for 16 hours at RT, formalin should be used in a fume hood |
| Glass histology slides- Ultra Super Frost, Menzel Glaze, 25×75 x1.0mm | TRAJAN | J3800AM4 | Using positive charged coated slides is essential. We do not recommend using poly-L-lysine for coating slides as tissue dislodges from slides during the antigen retrieval step |
| Goat anti human/mouse MPO antibody | R&D | AF3667 | Aliquot and freeze at minus 80 degrees upon arrival |
| Histosol | Clini Pure | CPL HISTOSOL 08 | Used neat, in 200ml staining rack containers, use in a fume hood |
| Hydrophobic pen | VECTOR Labs | H-400 | Use to draw circle around kidney tissue |
| Mouse anti human/mouse Peptidyl arginase 4 (PAD4) | ABCAM | ab128086 | Aliquot and freeze at minus 80 degrees upon arrival |
| Nikon C1 confocal scanning laser head attached to Nikon Ti-E inverted Microscope | Coherent Scientific | Aliquot and freeze at minus 80 degrees upon arrival | |
| Phosphate Buffered Saline | SIGMA | P38135 | 0.01M PB/0.09% NaCl Make up 5L at a time |
| Pressure Cooker 6L Tefal secure 5 Neo stainless | Tefal | GSA-P2530738 | Purchased at local homeware store |
| Prolong Gold DAPI | Life Technologies | P36962 | Apply drops directly to coverslip |
| Rabbit anti human/mouse Beta Actin antibody | ABCAM | ab8227 | Aliquot and freeze at minus 80 degrees upon arrival |
| Rabbit anti human/mouse H3Cit antibody | ABCAM | ab5103 | Aliquot and freeze at minus 80 degrees upon arrival |
| Staining rack 24 slides | ProScitech | H4465 | Staining rack chosen has to be able to withstand boiling under pressure and incubation in 60 degree oven |
| Sudan Black B | SIGMA | 199664 | 0.3% Add 3g to a 1L bottle in 70% Ethanol, filter and protect from the light- stable for 6 months at room temperature |
| Tris 10mM | SIGMA | T4661 | Add TRIS and EDTA together in distilled water and pH to 9, for antigen retrieval, can be made up in a 10x solution |
| Xylene | Trajan | XL005/20 | Must be use used in a fume hood |
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