一种同时取硬脑膜及颈深淋巴结的实验方法  

An experimental method for simultaneously extracting the dura mater and deep cervical lymph nodes

作　　者：谌子龙 吴铭杰 陈晓婧 周西彬 周春祥[1] Shen Zilong;Wu Mingjie;Chen Xiaojing;Zhou Xibin;Zhou Chunxiang(College of Traditional Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210000,Jiangsu Province,China)

机构地区：[1]南京中医药大学中医学院,江苏省南京市210000

出　　处：《中国组织工程研究》2025年第26期5543-5548,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家中医药管理局高水平中医药重点学科建设项目资助(国中医药人教函〔2023〕85号),项目负责人:周春祥;国家自然科学基金面上项目(82074504),项目负责人:周春祥;江苏省研究生科研与实践创新计划项目(KYCX24_2194),项目负责人:谌子龙。

摘　　要：背景:脑膜淋巴管可引流脑脊液和β-淀粉样蛋白,促进T淋巴细胞运输以及归巢至颈深淋巴结。简单快捷、步骤明确的硬脑膜分离方法以及颈深淋巴结的准确定位,可以为神经退行性疾病的研究提供有力的支持。目的:建立一种便捷实用的硬脑膜及颈深淋巴结剥取方法。方法:取3月龄ICR小鼠,麻醉后于枕大池注射埃文斯蓝和示踪剂进行颈深淋巴结定位,于锁骨上方约5 mm作长约3 cm的中线切口后,钝性分离浅脂肪和筋膜,牵拉侧胸锁乳突肌,暴露颈深淋巴结,在体式显微镜下摘取淋巴结,-80℃冻存;随后剪断小鼠颈部,分离头部皮肤肌肉暴露出完整颅骨结构,使用剪刀自枕骨大孔处沿顶骨下沿离断一周,分离颅骨和脑组织,获取完整的颅骨顶部。分别将取下来的颅骨顶部泡在40g/L多聚甲醛24h,120g/L多聚甲醛固定24h,120g/L多聚甲醛固定10,20,30,40min后,剥离硬脑膜结构。分别采用示踪剂对脑膜淋巴管、颈深淋巴引流能力进行验证,淋巴管内皮透明质酸受体1免疫荧光法鉴定脑膜淋巴管。结果与结论:①小鼠注射埃文斯蓝15 min后于颈深淋巴结内观察到明显蓝染。②颅骨置于120 g/L多聚甲醛固定24 h,可使硬脑膜与颅骨的连接不再紧密,剥离硬脑膜结构更为容易,且形状完整;与传统40 g/L多聚甲醛相比,120 g/L多聚甲醛固定的硬脑膜更具韧性,剥离时更能保持完整;120 g/L多聚甲醛短时间固定取脑膜以30-40 min较佳。③颈深淋巴结冰冻切片有明显示踪剂存在,硬脑膜可见完整的脑膜淋巴管存在,淋巴管尾端可见示踪剂;颈深淋巴结及硬脑膜淋巴管的淋巴管内皮透明质酸受体1免疫荧光染色均阳性。④结果说明,120 g/L多聚甲醛固定24 h为较佳的剥离浓度与时间,该浓度下硬脑膜形态舒展、韧性较好,剥离后较完整,利于后期使用;埃文斯蓝定位及示踪剂和免疫荧光验证的颈深淋巴结位置准确,可作为研究各�BACKGROUND:Meningeal lymphatic vessels can drain cerebral spinal fluid and amyloidβ-protein,promoting T lymphocyte to transport and home to deep cervical lymph nodes.A simple,quick and definite method of dural separation and accurate localization of deep cervical lymph nodes can provide strong support for the study of neurodegenerative diseases.OBJECTIVE:To establish a convenient and practical method for exfoliating dural and deep cervical lymph nodes.METHODS:ICR mice,3 months old,were taken,anesthetized and injected with Evans blue and tracer in the occipital pool for localizing deep cervical lymph nodes.A midline incision of about 3 cm in length was made about 5 mm above the clavicle,the superficial fat and fascia were bluntly separated,and the lateral sternocleidomastoid muscle was pulled to expose the deep cervical lymph nodes,which were removed under a stereomicroscope and frozen at-80°C.Subsequently,the mouse head was cut and the skin and muscles of the head were separated to expose the entire skull structure.The skull and brain tissue were separated from the foramen magnum along the lower parietal bone with scissors,and the complete skull top was obtained.The skull was sequentially fixed in 40 g/L paraformaldehyde solution for 24 hours,120 g/L paraformaldehyde for 24 hours,and 120 g/L paraformaldehyde for 10,20,30,and 40 minutes,and the dural structure was stripped.The drainage capacity of meningeal lymphatic vessels and deep cervical lymphatic vessels was verified by tracer,and the meningeal lymphatic vessels were identified by the lymphatic vessel endothelial hyaluronan receptor 1 using the immunofluorescence method.RESULTS AND CONCLUSION:(1)Obvious blue staining was observed in deep cervical lymph nodes 15 minutes after Evans blue staining.(2)The skull was sampled and fixed in 120 g/L paraformaldehyde for 24 hours,resulting in a less tight connection between the dura mater and the skull,and easier stripping of the dural structures with an intact shape.The dura mater fixed at 120 g/L concentration was

关 键 词：硬脑膜 颈深淋巴结 脑膜淋巴管 脑淋巴引流 阿尔茨海默 神经退行性疾病 工程化组织构建 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R446