原代大鼠主动脉血管干细胞的体外培养扩增及鉴定

doi:10.12122/j.issn.1673-4254.2024.10.06

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 1881-1886.doi: 10.12122/j.issn.1673-4254.2024.10.06

• • 上一篇

原代大鼠主动脉血管干细胞的体外培养扩增及鉴定

马华根¹^,²(), 黄燕², 杨盈欣², 刘海琴³, 唐元瑜⁴(), 丛伟红¹()

^1.中国中医科学院西苑医院，北京 100091
^2.北京中医药大学中医学院，北京 100029
^3.海军军医大学上海长征医院，上海 200003
^4.福建中医药大学中医学院，福建福州 350122

收稿日期:2024-03-11 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 唐元瑜,丛伟红 E-mail:mahuagen123666@163.com;2422198977@qq.com;congcao@188.com
作者简介:马华根，在读博士研究生，E-mail: mahuagen123666@163.com
基金资助:
国家自然科学基金(81973594)

Expansion and identification of primary rat aortic vascular stem cells in vitro

Huagen MA¹^,²(), Yan HUANG², Yingxin YANG², Haiqin LIU³, Yuanyu TANG⁴(), Weihong CONG¹()

^1.Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, China
^2.School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China
^3.Shanghai Changzheng Hospital, Naval Medical University, Shanghai 200003, China
^4.School of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, China

Received:2024-03-11 Online:2024-10-20 Published:2024-10-31
Contact: Yuanyu TANG, Weihong CONG E-mail:mahuagen123666@163.com;2422198977@qq.com;congcao@188.com
Supported by:
National Natural Science Foundation of China(81973594)

摘要/Abstract

摘要：

目的采用“切段外膜贴壁培养法”体外培养扩增出原代大鼠主动脉血管干细胞，为开展血管重构及相关疾病研究提供实用的工具细胞。方法无菌分离2~3周龄Sprague-Dawley大鼠胸腹主动脉，切成长约2.0 mm的血管节段，均匀种瓶，经外膜贴壁固化后进行原代培养，待细胞生长融合度达80%~90%时传代。镜下观察细胞形态及其生长特性，采用流式细胞仪分析目的细胞表面标志物CD分子表达情况，成脂、成骨诱导分化实验检测其多向分化潜能。结果接种3 d后，少量梭形、星形或多角形的细胞从血管节段周围爬出；5~6 d后形成岛屿状细胞集落，细胞增殖迅速，呈放射状向外扩大，并出现细胞克隆现象；7~8 d后细胞融合成片，呈涡旋状分布；传至第3代后，细胞匀质性较高，表现为典型的“成纤维样”排列生长。流式细胞仪分析细胞主要表达CD44、CD73、CD90，阳性率分别为80.3%、62.2%、46.8%；低表达CD34、CD45、CD11b/c，阳性率分别为1.1%、0.2%、0.2%。体外诱导分化实验表明目的细胞具有成脂、成骨分化潜能。结论 “切段外膜贴壁培养法”可成功分离培养出具有间充质干细胞特性的大鼠主动脉血管干细胞。

关键词: 血管干细胞, 主动脉, 切段外膜贴壁培养法, 原代培养, 免疫表型, 分化潜能, 血管重构

Abstract:

Objective To culture and expand primary rat aortic vascular stem cells in vitro and evaluate their characteristics as mesenchymal stem cells. Methods The thoracic and abdominal aortas isolated from 2- to 3-week-old Sprague-Dawley rats were cut into vascular segments 2.0 mm in length and cultured in culture flasks till adhesion and solidification of the outer membranes. The primary cells were further cultured to 80%-90% confluence before passaging. The morphology and growth characteristics of the cells were observed under a microscope, and the expressions of surface marker CD molecules on the cells was analyzed using flow cytometry. Adipogenic and osteogenic differentiation assays were performed to assess the capacity of the cells for multilineage differentiation. Results After 3 days of culture, a small number of spindle, star-shaped or polygonal cells migrated out from the peripheral of the vascular segments. At 5-6 days, island-like cell clusters occurred and the cells began to proliferate rapidly. The cell clusters expanded radially and showed signs of cell cloning. At 7-8 days, the cells fused into sheets and displayed a vortex-like distribution. The cells in the third passage presented with a uniform morphology, showing a typical fibroblast-like arrangement. Flow cytometry showed that the cells expressed predominantly CD44 (80.3%), CD73 (62.2%) and CD90 (46.8%) with low expressions of CD34 (1.1%), CD45 (0.2%) and CD11b/c (0.2%). Adipogenic and osteogenic differentiation experiments demonstrated that the cells were capable of lipogenic and osteogenic differentiation in vitro. Conclusion Rat aortic vascular stem cells with mesenchymal stem cell characteristics can be successfully isolated and cultured by adherent culture of the segmented outer membrane.

Key words: vascular stem cells, aorta, adherent culture of the segmented outer membrane, primary culture, immunophenotype, differentiation potential, vascular remodeling

马华根, 黄燕, 杨盈欣, 刘海琴, 唐元瑜, 丛伟红. 原代大鼠主动脉血管干细胞的体外培养扩增及鉴定[J]. 南方医科大学学报, 2024, 44(10): 1881-1886.

Huagen MA, Yan HUANG, Yingxin YANG, Haiqin LIU, Yuanyu TANG, Weihong CONG. Expansion and identification of primary rat aortic vascular stem cells in vitro[J]. Journal of Southern Medical University, 2024, 44(10): 1881-1886.

图/表 5

图1 原代主动脉VSCs的形态学观察

Fig.1 Morphological observation of rat aortic vascular stem cells (VSCs) in primary culture. A: Spindle-shaped, star-shaped or polygonal cells emerging from the peripheral of the vascular segments after 3 days of culture. B, C: After 5-6 days of culture, island-like cell colonies occurred and extended outward rapidly in a radial pattern. D: Cell cloning in the cell culture. E, F: After 7-8 days of culture, the cell density further increased, the cell colonies merged to exhibit a vortex-like distribution, and the cells formed a monolayer with adherent growth.

图2 P3~P8主动脉VSCs的形态学观察

Fig.2 Morphological observation of rat aortic VSCs in the third to eighth passages. A-C: After passaging for 4-6 h, the cells became adherent and spread uniformly, exhibiting a consistent morphology. The individual cells displayed a typical fibroblast-like appearance with pseudopodia extending from both poles of the cells. D-F: After 36-48 h, the passaged cells aligned tightly along the longitudinal axis, forming a spiral pattern with a clear orientation.

图3 主动脉VSCs表面标志物CD分子表达

Fig.3 Expression of surface marker CD molecules on cultured rat aortic VSCs. The positive rates of CD44, CD73, CD90, CD34, CD45 and CD11b/c were 80.3%, 62.2%, 46.8%, 1.1%, 0.2% and 0.2%, respectively.

图4 主动脉VSCs诱导成脂及油红O染色观察

Fig.4 Observation of induced adipogenesis of cultured rat aortic VSCs and oil red O staining of the cells. A, B: After 14 days of adipogenic induction, translucent lipid droplets appeared in the cytoplasm. C, D: Oil red O staining highlighting the bright red lipid droplets. E, F: Absence of translucent lipid droplets in the negative control cells before and after induction.

图5 主动脉VSCs诱导成骨及茜素红染色观察

Fig.5 Observation of induced osteogenesis of cultured rat aortic VSCs and alizarin red staining of the cells. A, B: After 21 days of osteogenic induction, irregular dense mineralized nodules occurred at the cell aggregations. C, D: Alizarin red staining showing bright red nodules. E, F: Absence of mineralized nodules in the negative control cells before and after induction.

参考文献 28

1	Lin CS, Lue TF. Defining vascular stem cells[J]. Stem Cells Dev, 2013, 22(7): 1018-26.
2	李诗媛, 周敏, 丁利军. 血管周围干细胞在组织纤维化和损伤修复中的作用[J]. 中国细胞生物学学报, 2023, 45(8): 1248-56.
3	Wörsdörfer P, Mekala SR, Bauer J, et al. The vascular adventitia: an endogenous, omnipresent source of stem cells in the body[J]. Pharmacol Ther, 2017, 171: 13-29.
4	Tang J, Wang HX, Huang XZ, et al. Arterial Sca1⁺ vascular stem cells generate de novo smooth muscle for artery repair and regeneration[J]. Cell Stem Cell, 2020, 26(1): 81-96. e4.
5	Zhang L, Issa Bhaloo S, Chen T, et al. Role of resident stem cells in vessel formation and arteriosclerosis[J]. Circ Res, 2018, 122(11): 1608-24.
6	Wang D, Li LK, Dai T, et al. Adult stem cells in vascular remodeling[J]. Theranostics, 2018, 8(3): 815-29.
7	Jiang LJ, Sun XL, Deng JC, et al. Different roles of stem/progenitor cells in vascular remodeling[J]. Antioxid Redox Signal, 2021, 35(3): 192-203.
8	Klein D, Weisshardt P, Kleff V, et al. Vascular wall-resident CD44⁺ multipotent stem cells give rise to pericytes and smooth muscle cells and contribute to new vessel maturation[J]. PLoS One, 2011, 6(5): e20540.
9	郭虹, 刘杰文, 杨少光, 等. 地塞米松促进胎儿主动脉血管CD105⁺间充质干细胞向脂肪细胞分化[J]. 中国病理生理杂志, 2004, 20(10): 1786-9. DOI: 10.3321/j.issn:1000-4718.2004.10.010
10	刘煜昊, 房佰俊, 史明霞, 等. 自发性高血压大鼠间充质干细胞生物学特性变化的研究[J]. 中国生物工程杂志, 2004, 24(04): 49-53. DOI: 10.3969/j.issn.1671-8135.2004.04.011
11	凌思英, 张柏杨, 滕勇, 等. 人大隐静脉干细胞原代培养方法的改良[J]. 生物工程学报, 2018, 34(6): 973-82. DOI: 10.13345/j.cjb.170459
12	杨礼菊, 马颖, 李源, 等. 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): 205-15.
13	Han CT, Xie C, Dang QY, et al. Immunomagnetic isolation of the vascular wall-resident CD34⁺ stem cells from mice[J]. J Vis Exp, 2023(202): e66193.
14	Wu Y, Zhang RN, Sen-Zhao, et al. Isolation and culture of vascular wall-resident CD34⁺ stem/progenitor cells[J]. Cardiology Plus, 2019, 4(4): 116-20.
15	唐元瑜, 刘海琴, 马华根. 原代SD大鼠主动脉内皮细胞培养方法的建立及其鉴定[J]. 吉林大学学报 (医学版), 2021, 47(04): 1038-42.
16	Lin SG, Lin RR, Zhang HK, et al. Peripheral vascular remodeling during ischemia[J]. Front Pharmacol, 2022, 13: 1078047.
17	Ye C, Zheng F, Wu N, et al. Extracellular vesicles in vascular remodeling[J]. Acta Pharmacol Sin, 2022, 43(9): 2191-201.
18	Hu Y, Zhang Z, Torsney E, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice[J]. J Clin Invest, 2004, 113(9): 1258-65.
19	李伟慷, 崔清卓, 郑玉光, 等. 基于TLR4/NF-κB/NLRP3通路的羟基红花黄色素A抑制血管紧张素II诱导的血管外膜成纤维细胞迁移研究[J]. 中草药, 2023, 54 (05): 1478-86. DOI: 10.7501/j.issn.0253-2670.2023.05.014
20	Klein D. Vascular wall-resident multipotent stem cells of mesenchymal nature within the process of vascular remodeling: cellular basis, clinical relevance, and implications for stem cell therapy[J]. Stem Cells Int, 2016, 2016: 1905846.
21	Tyurin-Kuzmin PA, Hayashi Y, Kulebyakin K. Editorial: functional heterogeneity of stem cells[J]. Front Cell Dev Biol, 2023, 11: 1179911.
22	Orekhov AN, Bobryshev YV, Chistiakov DA. The complexity of cell composition of the intima of large arteries: focus on pericyte-like cells[J]. Cardiovasc Res, 2014, 103(4): 438-51.
23	Narbonne P. The effect of age on stem cell function and utility for therapy[J]. Cell Med, 2018, 10: 2155179018773756.
24	李媛, 钟海英, 董世访, 等. 不同年龄小鼠骨来源间充质干细胞衰老相关特性与成骨分化能力的比较研究[J]. 陆军军医大学学报, 2024, 46 (13): 1512-22.
25	Greuel S, Hanci G, Böhme M, et al. Effect of inoculum density on human-induced pluripotent stem cell expansion in 3D bioreactors[J]. Cell Prolif, 2019, 52(4): e12604.
26	Liang L, Xu L, Dong Q, et al. Low initial cell density promotes the differentiation and maturation of human pluripotent stem cells into erythrocytes[J]. Stem Cells Dev, 2024, 33(11-12): 321-31.
27	Yang GQ, Fan XH, Liu YC, et al. Immunomodulatory mechanisms and therapeutic potential of mesenchymal stem cells[J]. Stem Cell Rev Rep, 2023, 19(5): 1214-31.
28	Wan XX, Hu XM, Xiong K. Multiple pretreatments can effectively improve the functionality of mesenchymal stem cells[J]. World J Stem Cells, 2024, 16(2): 58-63.

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原代大鼠主动脉血管干细胞的体外培养扩增及鉴定

Expansion and identification of primary rat aortic vascular stem cells in vitro

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