杨礼菊1,2, 马颖1, 李源1, 党青亚1, 程俊1, 杨艳1, 李鹏云1,*
1西南医科大学心血管医学研究所，医学电生理学教育部重点实验室，医学电生理四川省重点实验室，四川省心血管疾病防治协同创新中心，泸州 646000；2西南医科大学附属医院心内科，泸州 646000
血管壁干细胞(vascular wall-resident stem cells, VW-SCs)在维持血管正常功能以及损伤血管的修复过程中发挥着关键性的作用，对其功能特性的研究将有助于干细胞的调控及应用。本文旨在建立稳定的VW-SCs分离、培养及分选鉴定技术，为进一步深入研究VW-SCs在生理和疾病状态下的增殖、迁移和分化等机制提供丰富、可靠的细胞来源。首先利用组织块贴壁法获取小鼠主动脉外膜以及肠系膜动脉血管壁细胞，传代培养到细胞数量至少达到1 × 107后经磁珠分选和流式细胞术鉴定CD34+的VW-SCs；其次采用细胞免疫荧光染色检测干细胞标志物(CD34、Flk-1、c-kit、Sca-1)以及平滑肌标志物(SM22、SM MHC)、内皮标志物(CD31)和细胞核内分裂增殖相关蛋白(Ki-67)；血管壁CD34+干细胞的定向分化采用EBM-2内皮分化诱导培养基和FM-2成纤维细胞培养基分别培养7天和3天；利用细胞免疫荧光染色和q-PCR检测内皮细胞标志物(CD31, VWF)和成纤维细胞标志物(Vimentin, PDGFRα)的表达。此外，采用TILLvisION胞内钙信号检测系统结合Ca2+荧光探针Fura-2/AM观察CD34+干细胞胞内钙库Ca2+释放和胞外Ca2+内流特性。结果显示，血管组织块贴壁法和磁珠分选可获取较高纯度(> 90%)的小鼠主动脉外膜以及肠系膜动脉血管壁CD34+干细胞。体外培养的血管壁CD34+干细胞可诱导分化为内皮细胞和成纤维细胞。咖啡因和ATP可显著激活CD34+干细胞胞内钙库Ca2+释放，毒胡萝卜素(thapsigargin, TG)耗竭内质网钙库Ca2+后触发钙库操纵的外钙内流(store-operated Ca2+ entry, SOCE)。该方法建立了稳定、高效的血管壁CD34+干细胞分离、培养和鉴定技术，为体外深入研究VW-SCs的调控机制以及靶向药物的筛选提供了保障。
Isolation, culture and validation of CD34+ vascular wall-resident stem cells from mice
YANG Li-Ju1,2, MA Ying1, LI Yuan1, DANG Qing-Ya1, CHENG Jun1, YANG Yan1, LI Peng-Yun1,*
1Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, China；2Department of Cardiology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34+ VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34+ VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34+ VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca2+ release and extracellular Ca2+ entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34+ VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34+ VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca2+ release from endoplasmic reticulum of CD34+ VW-SCs. Store-operated Ca2+ entry (SOCE) was activated by using thapsigargin (TG) applied in Ca2+-free/Ca2+ reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34+ VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.
Key words: vascular wall-resident stem cells; cell isolation; cell identification; magnetic separation
通讯作者：李鹏云 E-mail: firstname.lastname@example.org
杨礼菊, 马颖, 李源, 党青亚, 程俊, 杨艳, 李鹏云. 小鼠血管壁CD34+干细胞的分离、培养及鉴定[J]. 生理学报 2023; 75 (2): 205-215.
YANG Li-Ju, MA Ying, LI Yuan, DANG Qing-Ya, CHENG Jun, YANG Yan, LI Peng-Yun. Isolation, culture and validation of CD34+ vascular wall-resident stem cells from mice. Acta Physiol Sin 2023; 75 (2): 205-215 (in Chinese with English abstract).