激光捕获显微切割从口腔粘膜下纤维化样本中分离具有形态学和空间信息的细胞
1Department of Oral Pathology,Peking University School and Hospital of Stomatology & National Center of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, 2Research Unit of Precision Pathologic Diagnosis in Tumors of the Oral and Maxillofacial Regions,Chinese Academy of Medical Sciences (2019RU034), 3Central Laboratory,Peking University School and Hospital of Stomatology
Summary
口腔粘膜下纤维化组织的激光捕获显微切割允许从感兴趣的组织学区域精确提取细胞，以分析具有形态学和空间信息的多组学数据。
Abstract
口腔粘膜下纤维化 （OSF） 是口腔中一种常见的潜在恶性疾病。上皮萎缩以及固有层和粘膜下层的纤维化经常在组织病理学载玻片上发现。上皮发育不良、上皮萎缩和衰老成纤维细胞被认为与 OSF 的恶性转化有关。然而，由于潜在恶性口腔疾病和口腔鳞状细胞癌的异质性，很难确定OSF中恶性转化的具体分子机制。在这里，我们提出了一种通过在福尔马林固定石蜡包埋的组织载玻片上进行激光捕获显微切割来获得少量携带形态学数据和空间信息的上皮或间充质细胞的方法。使用显微镜，我们可以精确捕获微尺度（~500 个细胞）发育不良或萎缩的上皮组织和纤维化上皮下组织。提取的细胞可以通过基因组或转录组测序进行评估，以获取具有形态学和空间学信息的基因组和转录组数据。这种方法消除了大量OSF组织测序的异质性和非病变区域细胞引起的干扰，从而可以对OSF组织进行精确的空间组学分析。
Introduction
口腔粘膜下纤维化 （OSF） 是一种慢性隐匿性疾病，主要发生在颊粘膜，导致张口受限1。虽然 OSF 是一种多因素疾病，但槟榔或咀嚼槟榔是 OSF的主要原因 2,3。由于这种地理上特定的习性，OSF主要集中在东南亚和南亚的人群中3。OSF 的常见组织学特征包括口腔黏膜上皮下方结缔组织中胶原蛋白异常沉积、血管狭窄和闭塞1。OSF上皮组织可表现为萎缩或增生，甚至在口腔白斑时出现异型增生4,5。
OSF 被世界卫生组织定义为一种常见的口腔潜在恶性疾病 （OPMD），具有进展为口腔鳞状细胞癌的潜力，恶性转化率为 4%-6%6,7,8,9。OSF恶性转化的机制是复杂的10。上皮细胞的异常生长，包括发育不良和萎缩，增加了致癌的可能性，基质中的衰老成纤维细胞可能通过活性氧 （ROS） 和其他分子诱导上皮-间充质转化 （EMT） 参与 OSF 的恶性进展10。
空间组学分析技术生成了具有形态学和空间信息的多组学数据，为癌症机制提供了见解11,12,13。在这里，我们提出了一种通过激光显微切割从福尔马林固定石蜡包埋的OSF组织中捕获形态相关细胞群的方案。对这些样本进行多组学分析可以克服组织内异质性的挑战，并增加对 OSF14 中分子病理学和恶性转化机制的理解。
Protocol
本研究经北京大学附属学校机构评审委员会批准。已获得患者的知情同意。本研究中使用的组织样本被去标识化。研究方案如图 1所示。 1. 样品制备 在切片机上将福尔马林固定石蜡包埋的口腔粘膜下纤维化组织切成3μm和10μm厚度的连续切片。 在水中展开各部分，然后捞到载玻片上。将3μM和10μm切片分别固定在粘附显微镜载玻…
Representative Results
通过对OSF组织进行激光显微切割，我们捕获了发育不良上皮、发育不良上皮下的基质、萎缩上皮和萎缩上皮组织下的基质样本（图1）。通过提取DNA和低深度全基因组测序，我们能够分析形态学相关的拷贝数改变（CNA）15。CNA 是一种常见的基因组不稳定形式，与 OPMD15,16 中恶性转化的风险增加有关。我们在4种样本?…
Discussion
该协议报告了一个管道，用于捕获具有形态和空间信息的OSF组织样本，以便通过激光显微切割进行进一步的空间组学分析。从代表性结果中，我们在各种形态相关样品中鉴定出不同的CNA模式。
OSF 是 OPMD 的一种，是口腔鳞状细胞癌6 的常见癌前病变。据报道，基因组不稳定性与 OPMD17,18 的发展和恶性转化有关。多项?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究得到了国家自然科学基金（81671006、81300894）、中国医学科学院医学创新基金（2019-I2M-5-038）、国家临床重点学科建设项目（PKUSSNKP-202102）、北京大学医学部优秀博士生创新基金（BMU2022BSS001）的资助。
Materials
| Adhesion microscope slides | CITOTEST | REF.188105 | |
| Div-haematoxylin | YiLi | 20230326 | |
| Eosin solution | BASO | BA4098 | |
| Ethanol | PEKING REAGENT | No.32061 | |
| Harris hematoxylin dye solution | YiLi | 20230326 | |
| Hot plate | LEICA | HI1220 | |
| Laser capture microdissection system | LEICA | LMD7 | Machine |
| Laser microdissection microsystem | LEICA | 8.2.3.7603 | Software |
| Micromount mounting medium | LEICA | REF.3801731 | |
| Microscope cover glass | CITOTEST | REF.10212450C | |
| Microtome | LEICA | RM2235 | |
| PCR tubes | AXYGEN | 16421959 | |
| PEN-membrane slides | LEICA | No.11505158 | |
| Re-blue solution | YiLi | 20230326 | |
| Ultrapure distilled water | Invitrogen | REF.10977-015 | |
| Xylene | PEKING REAGENT | No.33535 |
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Cite This Article
Cai, X., Zhang, H., Zhang, J., Li, T. Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection. J. Vis. Exp. (198), e65890, doi:10.3791/65890 (2023).