从人骨关节炎膝关节收集和RNA提取
Summary
从全膝关节置换术后的患者获得的原代组织为骨关节炎研究提供了一个实验模型，具有最大的临床可转化性。该协议描述了如何从七个独特的膝关节组织中鉴定，处理和分离RNA，以支持人类骨关节炎的机制研究。
Abstract
骨关节炎（OA）是一种慢性退行性关节疾病，最常影响膝盖。由于目前尚无治愈方法，全膝关节置换术（TKA）是一种常见的手术干预。使用从TKA获得的原代人OA组织的实验提供了研究 离体疾病机制的能力。虽然OA以前被认为主要影响软骨，但现在已知它会影响关节中的多个组织。该协议描述了来自七个独特组织的患者选择，样品处理，组织均质化，RNA提取和质量控制（基于RNA纯度，完整性和产量），以支持膝关节的疾病机制研究。经知情同意，从接受TKA治疗OA的患者处获得样本。在手术后4小时内通过闪光冷冻RNA或福尔马林固定进行组织学检查，洗涤和储存组织组织。收集的组织包括关节软骨、软骨下骨、半月板、髌下脂肪垫、前交叉韧带、滑膜和内侧斜肌。针对每种组织类型测试RNA提取方案。最显著的修饰涉及用于低细胞、高基质、硬组织（被视为软骨、骨骼和半月板）与相对高细胞、低基质、软组织（被视为脂肪垫、韧带、滑膜和肌肉）的崩解方法。研究发现，粉碎适用于硬组织，均质化适用于软组织。观察到一些受试者倾向于在多个组织中产生比其他受试者更高的RNA完整性数（RIN）值，这表明疾病严重程度等潜在因素可能会影响RNA质量。从原代人类OA组织中分离高质量RNA的能力为复杂的基因表达实验（包括测序）提供了生理相关模型，可以导致更容易转化为患者的临床见解。
Introduction
膝关节是人体最大的滑膜关节，包括胫骨和股骨之间的胫股关节以及髌骨和股骨之间的髌股关节1。膝盖中的骨骼衬有关节软骨，并由各种结缔组织支撑，包括半月板，脂肪，韧带和肌肉，滑膜封装整个关节以形成充满滑液的腔1，2，3（图1）。健康的膝盖作为一个移动的铰链关节，允许在额平面1，3中无摩擦运动。在病理条件下，运动会受到限制和疼痛。最常见的退行性膝关节疾病是骨关节炎（OA）4。已知各种危险因素易发生OA发展，包括年龄较大，肥胖，女性，关节创伤和遗传学，除其他外5，6。目前，美国估计有1400万人患有有症状的膝关节OA，由于人口年龄和肥胖率上升，患病率增加7，8。OA最初被认为是软骨的疾病，现在被理解为整个关节的疾病9。OA中通常观察到的病理变化包括关节软骨侵蚀，骨赘形成，软骨下骨增厚和滑膜炎症9，10。由于OA没有已知的治愈方法，治疗主要集中在症状（例如，疼痛）管理11，12，并且一旦OA进展到终末期，通常需要关节置换手术13。
关节置换手术可以是部分或全膝关节置换术，全膝关节置换术（TKA）包括置换整个胫股关节和髌股关节。截至2020年，美国每年约有100万个TKA进行14。在TKA期间，整形外科医生切除胫骨平台的上部和下股骨髁（图2A，2B）以安装假体植入物。有时被患者误解，在TKA中，只有8-10毫米从每块骨头的末端切除，随后用金属覆盖或重新表面。插入的聚乙烯衬垫在两个金属植入物之间形成轴承表面（即衬垫）。此外，关节的几个软组织成分被完全或部分切除，以实现适当的关节平衡。这些组织包括内侧和外侧半月板（图2C），髌下脂肪垫（图2D），前交叉韧带（ACL;图2E）、滑膜（图2F）和内侧斜肌（VMO;图2G）虽然TKA通常对OA治疗是成功的，但大约20%的患者报告手术后疼痛复发16。除了手术的高成本和相对侵入性外，这些局限性表明需要进一步研究以确定替代疗法以减轻OA的进展。
为了探索OA中的疾病机制，这些机制可能为治疗干预提供新的途径，可以使用实验系统，包括细胞，组织外植体和动物模型。细胞通常在单层中培养，并且来源于原代人或动物组织（例如，从软骨中分离的软骨细胞）或永生化细胞（例如，ATDC517 和CHON-00118）。虽然细胞可用于在受控培养环境中操纵实验变量，但它们不能捕获已知影响细胞表型的自然关节条件19。为了更好地概括OA背后的化学，机械和细胞间通讯的复杂级联反应，在原代人或动物组织样品中发现了一种替代方案，无论是使用新鲜还是 培养的离体 作为外植体，以保留组织结构和细胞微环境20。为了研究 体内关节，小（如小鼠21）和大（如马22）动物模型对OA（如手术诱导、遗传改变或衰老）也是有用的。然而，从这些模型到人类疾病的转化可能受到解剖学，生理学和代谢差异的限制，其中包括23。考虑到实验系统的优缺点，物种特异性和维持主要人类OA组织提供的细胞外生态位的关键优势最大限度地发挥了研究结果的转化潜力。
原代人类OA组织在TKA之后可以很容易地获得，这使得TKA的高频率成为研究的宝贵资源。潜在的实验应用包括基因表达和组织学分析。为了实现初级人类OA组织在这些研究方法和其他方面的潜力，概述了以下关键考虑因素。首先，患者标本的使用受道德规范的约束，并且协议必须符合机构审查委员会（IRB）的批准24。其次，人类原发性病变组织固有的异质性以及年龄和性别等变量的影响，使得需要仔细选择患者（即，应用资格标准）和数据解释。第三，关节中不同组织的独特生物学特性（例如，软骨和半月板25的低细胞性）在实验过程中可能会带来挑战（例如，分离高质量和数量的RNA）。本报告解决了这些考虑因素，并提出了患者选择，样品处理，组织均质化，RNA提取和质量控制（即评估RNA纯度和完整性; 图 3）鼓励在研究界使用初级人类OA组织。
Protocol
Representative Results
Discussion
所提出的方案已被证明可以成功地收集七种主要的人类OA组织进行RNA提取（表1）和组织学处理（图4）。在收集患者样本之前，有必要建立IRB批准的方案，最好是与外科医生或手术团队合作。应用标准化的实验方案进行标本采集（例如，从一致的原位位置切除）对于最大限度地提高实验再现性至关重要。组织样本应装在无菌容器中运输到实验室，并在手术?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者感谢使这项研究成为可能的研究参与者，并将本报告献给骨关节炎领域的新科学家。
Materials
| 1.5 mL microcentrifuge tubes | Eppendorf | 05 402 | Sterile, nuclease-free. Reserved for RNA work only. |
| 10% Formalin | Cardinal Health | C4320-101 | Store in chemical cabinet when not in use. |
| 100% Chloroform (Molecular Biology Grade) | Fisher Scientific | ICN19400290 | Sterile, nuclease-free. Reserved for RNA work only, store in chemical cabinet when not in use. |
| 100% Ethanol (Molecular Biology Grade) | Fisher Scientific | BP2818500 | Sterile, nuclease-free. Reserved for RNA work only, when diluting use DEPC/nuclease-free water. |
| 100% Isopropanol (Molecular Biology Grade) | Fisher Scientific | AC327272500 | Sterile, nuclease-free. Reserved for RNA work only, store in chemical cabinet when not in use. |
| 100% Reagent Alcohol | Cardinal Health | C4305 | Diluted to 70% with dH2O for cleaning purposes. |
| 15 cm sterile culture dishes | Thermo Scientific | 12-556-003 | Sterile, nuclease-free. |
| 15 mL polypropylene (Falcon) tubes | Fisher Scientific | 14 959 53A | Sterile, nuclease-free. |
| 2 mL cryovials (externally threaded) | Fisher Scientific | 10 500 26 | Sterile, nuclease-free. |
| 5 mL round-bottom tubes | Corning | 352052 | Sterile, nuclease-free. Reserved for RNA work only. |
| 50 mL polypropylene (Falcon) tubes | Fisher Scientific | 12 565 271 | Sterile, nuclease-free. |
| Bioanalyzer | Agilent | G2939BA | For RNA integrity measurement. |
| Biosafety Cabinet | General lab equipment | ||
| Bone Cutters | Fisher Scientific | 08 990 | Sterilized with 70% EtOH. |
| Chemical Fume Hood | General lab equipment | ||
| Disposable Scalpels (No.10) | Thermo Scientific | 3120032 | Sterile, nuclease-free. |
| EDTA | Life Technologies | 15-576-028 | 10% solution with dH2O. |
| Forceps | Any vendor | Sterilized with 70% EtOH. | |
| Glycoblue Coprecipitant | Fisher Scientific | AM9516 | Reserved for RNA work only, store at -20 °C. |
| Kimwipes | Fisher Scientific | 06-666 | |
| Liquid Nitrogen | Any vendor | ||
| Liquid Nitrogen Dewar | General lab equipment | ||
| Mortar and Pestle | Any vendor | Reserved for RNA work only, sterilzed per protocol. | |
| Nanodrop Spectrophotometer | Thermo Scientific | ND-2000 | For RNA purity and yield measurements. |
| Nuclease-free/DEPC-treated water | Fisher Scientific | Sterile, nuclease-free. Reserved for RNA work only. | |
| PBS (Sterile) | Gibco | 20 012 050 | Sterile, nuclease-free. |
| Pipettes (2 µL, 20 µL, 200 µL, 1000 µL) & tips | Any vendor | Sterile, nuclease-free. | |
| Plasma/Serum Advanced miRNA kit | Qiagen | 217204 | |
| Refrigerated Centrifuge 5810R | Eppendorf | 22625101 | |
| RNAlater | Thermo Scientific | 50 197 8158 | Sterile, nuclease-free. |
| RNAse Away/RNAseZap | Fisher Scientific | 7002 |
|
| Spatula (semimicro size) | Any vendor | Reserved for RNA work only. | |
| Tissue homogenizer | Pro Scientific | 01-01200 | Reserved for RNA work only, sterilzed per protocol. |
| TRIzol Reagent | Fisher Scientific | 15 596 026 | Sterile, nuclease-free. Reserved for RNA work only. |
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