摘要
本研究旨在体外分离培养出原代家兔脊髓微血管内皮细胞,为脊髓损伤研究提供实用的受试细胞源。无菌提取1月龄家兔脊髓组织,依次经剪碎、牛血清白蛋白密度梯度离心、筛网过滤、Ⅱ型胶原酶消化获得纯净的脊髓微血管段,将其与适量M199完全培养基混匀后接种于培养皿中进行原代培养;期间持续观察记录细胞的生长状况,并采用免疫组化检测其Ⅷ因子相关抗原表达情况。结果显示:脊髓微血管段接种24 h后,有少量内皮样细胞爬出;36~60 h细胞集落逐渐扩大并相互融合;72 h后细胞铺满皿底,呈“鹅卵石样”单层贴壁生长。免疫组化检测Ⅷ因子相关抗原,99%以上的细胞胞质染成棕红色,为阳性表达。上述结果表明,本研究成功建立了一种便捷、稳定的原代家兔脊髓微血管内皮细胞培养方法。
This study aims to isolate and culture primary rabbit spinal cord microvascular endothelial cells in vitro,providing a practical source of test cells for spinal cord injury research.Spinal cord tissue was aseptically extracted from one-month-old rabbits and processed sequentially through mincing,bovine serum albumin density gradient centrifugation,mesh filtration,and typeⅡcollagenase digestion to obtain purified spinal cord microvascular segments.The microvascular segments were homogeneously mixed with an apprapriate volume of M199 complete culture medium and seeded into a culture dish for primary culture.Throughout the culture period,cell growth performance were continuously observed and recorded.Additionally,immunocytochemical staining was performed to evaluate the expression of factorⅧ-related antigen.The results showed that after 24 hours of inoculation,a small amount of endothelial-like cells were observed to emerge from the spinal cord microvascular segments.Within 36~60 hours,the cell colonies gradually expanded and fused.After 72 hours,the cells spread across the base of the dish,forming a“cobblestone-like”monolayer.Immunocytochemical staining showed that more than 99%of the cells showed brown-red cytoplasm and were positive for factorⅧ-related antigen.It is these results that suggest this study has successfully established a convenient and stable primary rabbit spinal cord microvascular endothelial cells culture method.
作者
马华根
池茗
林芷伊
唐元瑜
丛伟红
MA Hua-Gen;CHI Ming;LIN Zhi-Yi;TANG Yuan-Yu;CONG Wei-Hong(Laboratory of Cardiovascular Diseases,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;College of Integrated Traditional Chinese and Western Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China;Teaching and Research Office of Basic Theory of Traditional Chinese Medicine,College of Traditional Chinese Medicine,Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)
出处
《中国生物化学与分子生物学报》
北大核心
2025年第7期1062-1067,共6页
Chinese Journal of Biochemistry and Molecular Biology
基金
中国中医科学院西苑医院能力提升项目(No.XYZX0303-02)
中医药传承与创新“百千万”人才项目(第四批全国西医学习中医优秀人才研修项目)(No.国中医药人教发[2019]13号)
国家自然科学基金(No.81072714)资助。
关键词
脊髓微血管内皮细胞
原代培养
Ⅷ因子相关抗原
家兔
脊髓损伤
spinal cord microvascular endothelial cells(SCMECs)
primary culture
factorⅧ-related antigen
rabbit
spinal cord injury
