中华皮肤科杂志 ›› 2025, Vol. 58 ›› Issue (5): 453-459.doi: 10.35541/cjd.20240400

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卡波西样血管内皮瘤来源内皮细胞的分离、纯化、鉴定、培养与表型分析

兰钰茹1    周江元1    邱桐1    龚雪1    杨开颖2    张梓欣1    张学鹏3    吉毅1   

  1. 1四川大学华西医院小儿外科，成都  610041；2广州医科大学附属妇女儿童医疗中心小儿外科，广州  510623；3四川大学华西医院重症医学科，成都  610041
  • 收稿日期:2024-07-29 修回日期:2024-10-30 发布日期:2025-04-30
  • 通讯作者: 吉毅 E-mail:jijiyuanyuan@163.com
  • 基金资助:
    国家自然科学基金（82273556、82473553）；四川省科技厅重点研发项目（2022YFS0233、2022YFS0225 ）；四川大学从0到1创新研究项目（2022SCUH0033）；四川大学医学+信息中心交叉学科建设开放项目（YGJC004）；四川大学华西医院学科卓越发展1·3·5工程临床研究孵化项目（2019HXFH056、2020HXFH048、2023HXFH004）

Isolation, purification, identification, culture, and phenotypic analysis of endothelial cells derived from Kaposiform hemangioendothelioma

Lan Yuru1, Zhou Jiangyuan1, Qiu Tong1, Gong Xue1, Yang Kaiying2, Zhang Zixin1, Zhang Xuepeng3, Ji Yi1   

  1. 1Department of Pediatric Surgery, West China Hospital, Sichuan University, Chengdu 610041, China; 2Department of Pediatric Surgery, Guangzhou Women and Children′s Medical Center, Guangzhou Medical University, Guangzhou 510623, China; 3Department of Critical Care Medicine, West China Hospital, Sichuan University, Chengdu 610041, China
  • Received:2024-07-29 Revised:2024-10-30 Published:2025-04-30
  • Contact: Ji Yi E-mail:jijiyuanyuan@163.com
  • Supported by:
    National Natural Science Foundation of China （82273556, 82473553）; Key Project in the Science & Technology Program of Sichuan Province （2022YFS0233, 2022YFS0225）; Project of ‘0 to 1′ of Sichuan University （2022SCUH0033）; Med-X Center for Informatics Funding Project （YGJC004）; 1·3·5 Project for Disciplines of Excellence-Clinical Research Incubation Project of West China Hospital of Sichuan University （2019HXFH056, 2020HXFH048, 2023HXFH004）

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摘要： 【摘要】 目的 建立卡波西样血管内皮瘤（KHE）来源内皮细胞（KHE-EC）分离、纯化、鉴定、培养的完整体系，分析KHE-EC的表型，探讨建立KHE-EC库的可能性。方法 用收集液、LiberaseTM原液和分散酶原液配制一种全新KHE瘤体消化液（专利号: CN202410500224.2），用该消化液处理瘤体以获取细胞。运用CD31+免疫磁珠法纯化获得高纯度KHE-EC。EGM-2完全培养基（含10%胎牛血清和2%青霉素-链霉素溶液）用于细胞培养。为验证KHE-EC细胞特性，采用免疫荧光检测内皮细胞特异性标志物CD31、CD34，KHE疾病标志物D2-40、Prospero相关同源异形盒蛋白1（Prox-1）、淋巴管内皮透明质酸受体1（LYVE1）和婴幼儿血管瘤特异性诊断标志物葡萄糖转运蛋白1（GLUT-1）的表达。使用人脐静脉内皮细胞（HUVEC）为对照，对KHE-EC进行细胞表型分析，包括细胞活力、细胞骨架、增殖、迁移、侵袭、成管和出芽能力。结果 成功从KHE瘤体组织中分离出原代细胞，通过CD31+免疫磁珠法获得了高纯度KHE-EC，细胞形态为典型的纺锤形，贴壁生长。免疫荧光检测结果显示，KHE-EC表达CD31、CD34、D2-40、Prox-1及LYVE1，不表达GLUT-1。KHE-EC与HUVEC的细胞形态、细胞活性、细胞骨架有明显差异，且KHE-EC细胞增殖百分比（29.1% ± 2.5%）、迁移（114.3 ± 9.4）和侵袭（110.0 ± 6.1）细胞数量、成管长度［（32 121.0 ± 892.0）μm］、出芽个数（25.0 ± 3.6）均明显高于HUVEC［13.0% ± 2.2%、38.0 ± 3.6、35.3 ± 2.3、（25 345.0 ± 448.1）μm、5.0 ± 1.0］，差异有统计学意义（均P ≤ 0.001）。结论 首次创新性地配制了一种针对KHE瘤体的消化液，结合CD31+免疫磁珠法成功分离并纯化了多株高纯度且生长良好的KHE-EC，为KHE的后续实验研究提供了稳定且可靠的细胞来源，也为建立KHE-EC细胞库奠定了基础。

关键词: 血管内皮瘤, 内皮细胞, 原代细胞培养, 卡波西样血管内皮瘤, 消化液

Abstract: 【Abstract】 Objective To establish a complete system for the isolation, purification, identification, and culture of Kaposiform hemangioendothelioma-derived endothelial cells （KHE-ECs）, to analyze the phenotype of KHE-ECs, and to explore the possibility of establishing a KHE-EC bank. Methods A novel digestion solution for KHE tumors （patent number: CN202410500224.2） was formulated using collection fluid, LiberaseTM and dispase stock solutions, and was used to process tumor tissues to obtain cells. High-purity KHE-ECs were purified using CD31+ immunomagnetic beads. The EGM-2 complete medium containing 10% fetal bovine serum and 2% penicillin-streptomycin solution was employed for cell culture. To verify the characteristics of KHE-ECs, immunofluorescence assay was conducted to determine the expression of endothelial cell-specific markers CD31 and CD34, KHE disease markers podoplanin （D2-40）, prospero-related homeobox 1 （Prox-1）, and lymphatic vessel endothelial hyaluronan receptor 1 （LYVE1）, as well as an infantile hemangioma-specific diagnostic marker glucose transporter 1 （GLUT-1）. Human umbilical vein endothelial cells （HUVECs） served as controls for the phenotype analysis of KHE-ECs, including cell viability, cytoskeleton, proliferation, migration, invasion, tube formation, and sprouting ability. Results Primary cells were successfully isolated from KHE tumor tissues, and high-purity KHE-ECs were obtained by using CD31+ immunomagnetic beads. The cells exhibited typical spindle-shaped morphology and an adherent growth pattern. Immunofluorescence assay showed that KHE-ECs expressed CD31, CD34, D2-40, Prox-1, and LYVE1, but did not express GLUT-1. There were significant differences in cell morphology, cell viability, and cytoskeletal structures between KHE-ECs and HUVECs. Additionally, the KHE-EC group showed significantly increased percentages of proliferative cells （29.1% ± 2.5%）, numbers of migratory cells （114.3 ± 9.4） and invasive cells （110.0 ± 6.1）, tube length （32 121.0 ± 892.0 μm）, and number of sprouting cells （25.0 ± 3.6） compared with the HUVEC group （13.0% ± 2.2%, 38.0 ± 3.6, 35.3 ± 2.3, 25 345.0 ± 448.1 μm, 5.0 ± 1.0, respectively, all P ≤ 0.001）. Conclusion An innovative digestion solution specifically for KHE tumors was formulated for the first time, and high-purity and well-growing KHE-EC strains were successfully isolated and purified by using the novel digestion solution in combination with CD31+ immunomagnetic beads, providing a stable and reliable cell source for subsequent experimental studies on KHE and laying the foundation for establishing a KHE-EC bank.

Key words: Hemangioendothelioma, Endothelial cells, Primary cell culture, Kaposiform hemangio-endothelioma, Digestive fluid

引用本文

兰钰茹 周江元 邱桐 龚雪 杨开颖 张梓欣 张学鹏 吉毅. 卡波西样血管内皮瘤来源内皮细胞的分离、纯化、鉴定、培养与表型分析[J]. 中华皮肤科杂志, 2025,58(5):453-459. doi:10.35541/cjd.20240400

Lan Yuru, Zhou Jiangyuan, Qiu Tong, Gong Xue, Yang Kaiying, Zhang Zixin, Zhang Xuepeng, Ji Yi. Isolation, purification, identification, culture, and phenotypic analysis of endothelial cells derived from Kaposiform hemangioendothelioma[J]. Chinese Journal of Dermatology, 2025, 58(5): 453-459.doi:10.35541/cjd.20240400