中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (1): 58-64.doi: 10.12307/2024.738

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

黑枸杞花青素联合人脂肪源性血管外膜细胞支持脐血造血干/祖细胞的增殖

申娅媚1，2，牛云霞1，杨婷婷3，马  洁4，胡代宏4，郑  波4   

  1. 1宁夏医科大学临床医学院，宁夏回族自治区银川市   750004；2宁夏回族自治区干细胞与再生医学重点实验室，宁夏回族自治区银川市   750004；宁夏医科大学总医院，3宁夏神经系统疾病诊疗工程技术研究中心，4血液内科，宁夏回族自治区银川市   750004
  • 收稿日期:2023-10-23 接受日期:2023-11-27 出版日期:2025-01-08 发布日期:2024-05-18
  • 通讯作者: 郑波，主任医师，教授，硕士生导师，宁夏医科大学总医院血液科，宁夏回族自治区银川市 750004
  • 作者简介:申娅媚，女，1989年生，甘肃省武威市人，汉族，宁夏医科大学在读硕士，主治医师，主要从事造血微环境与造血干细胞相关研究。
  • 基金资助:
    国家自然科学基金项目(81860028)，项目负责人：郑波；宁夏自然科学基金项目(2023AAC03527)，项目负责人：郑波

Anthocyanins from Lycium ruthenicum Murr combined with human adipose-derived pericytes/perivascular cells support proliferation of umbilical cord blood hematopoietic stem/progenitor cells

Shen Yamei1, 2, Niu Yunxia1, Yang Tingting3, Ma Jie4, Hu Daihong4, Zheng Bo4   

  1. 1Clinical Medicine College, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 2Ningxia Key Laboratory of Stem Cell and Regenerative Medicine, Yinchuan 750004, Ningxia Hui Autonomous Region, China; 3Diagnosis and Treatment Engineering Technology Research Center of Nervous Disease of Ningxia, 4Department of Hematology, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China 
  • Received:2023-10-23 Accepted:2023-11-27 Online:2025-01-08 Published:2024-05-18
  • Contact: Zheng Bo, Chief physician, Professor, Master’s supervisor, Department of Hematology, General Hospital of Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • About author:Shen Yamei, Master candidate, Attending physician, Clinical Medicine College, Ningxia Medical University, Yinchuan 750004, Ningxia Hui Autonomous Region, China; Ningxia Key Laboratory of Stem Cell and Regenerative Medicine, Yinchuan 750004, Ningxia Hui Autonomous Region, China
  • Supported by:
    National Natural Science Foundation of China, No. 81860028 (to ZB); Ningxia Natural Science Foundation Project, No. 2023AAC03527 (to ZB)

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摘要：

文题释义：
人脂肪源性血管外膜细胞：是骨髓间充质干细胞的前体细胞，表达CD146和间充质干细胞表面抗原CD105、CD90、CD73及CD44，具有自我更新能力和生血管潜能，在特定条件下能够向成骨、成脂和成软骨分化，并分泌高水平的蛋白质，在血管生成、血运重建、皮肤伤口愈合和组织再生中发挥重要作用。
脐血造血干/祖细胞：是异基因造血干细胞移植中骨髓造血干细胞的替代来源，异体排斥反应小，免疫原性低，但存在单份脐血造血干/祖细胞数量相对较少的局限性。近年来间充质干细胞和细胞因子等被用于体外扩增脐血造血干/祖细胞。


背景：黑枸杞花青素(Anthocyanins in Lycium ruthenicum Murr，ALRM)是黑枸杞中重要的活性成分之一，具有抗氧化、免疫调节等功效。人脂肪源性血管外膜细胞(CD146+hAD-PCs)是骨髓间充质干细胞的前体细胞，在体外具有促进造血干/祖细胞增殖与分化的功能。ALRM联合CD146+hAD-PCs对脐血造血干/组细胞的体外支持作用有待于研究。
目的：探讨ALRM联合CD146+hAD-PCs对脐血CD34+造血干/祖细胞体外扩增的支持作用。
方法：CCK-8法检测不同质量浓度ALRM(0，200，400，600，800，1 000 mg/L)对CD146+hAD-PCs增殖的影响；流式细胞术检测ALRM对CD146+hAD-PCs细胞周期的影响。共培养实验分为空白组、ALRM组、CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组，分析ALRM联合CD146+hAD-PCs对脐血CD34+造血干/祖细胞的体外支持作用。共培养1，2，4周，比较扩增后细胞数量、集落形成单位数量，流式细胞仪检测细胞免疫表型，ELISA检测细胞因子水平。
结果与结论：①ALRM质量浓度为200 mg/L时，CD146+hAD-PCs活力最高，CD146+hAD-PCs的G0/G1期细胞比例下降，S期、G2/M期细胞比例上升(P < 0.01)。②脐血CD34+造血干/祖细胞数量变化：在共培养1，2，4周时ALRM+CD146+hAD-PCs组高于ALRM组(P均 < 0.05)，在共培养2，4周时ALRM+CD146+hAD-PCs组高于CD146+hAD-PCs组(P均 < 0.05)，ALRM组与空白组随着共培养时间延长细胞数量逐渐减少。③集落形成能力及免疫表型分析：在共培养1，2周时ALRM+CD146+hAD-PCs组的集落形成单位数量高于CD146+hAD-PCs组和ALRM组(P均 < 0.05)；在共培养1，2，4周时ALRM+CD146+hAD-PCs组CD45+、CD34+CD33-细胞比例高于CD146+hAD-PCs组(P均 < 0.01)。④细胞因子变化：在共培养4周时ALRM+CD146+hAD-PCs组的白细胞介素2水平高于ALRM组、CD146+hAD-PCs组(P < 0.05)；在共培养2，4周时ALRM+CD146+hAD-PCs组白细胞介素3水平高于CD146+hAD-PCs组(P < 0.05)；在共培养1周时ALRM+CD146+hAD-PCs组的粒细胞集落刺激因子水平高于CD146+hAD-PCs组，在共培养2周时高于ALRM组、CD146+hAD-PCs组(P < 0.01)；在共培养1，2，4周时ALRM组、ALRM+CD146+hAD-PCs组的干扰素γ水平低于CD146+hAD-PCs组(P < 0.05)。⑤由于空白组无基质细胞，脐血CD34+造血干/祖细胞在共培养1周之后就无法计数，未进行免疫表型、集落分析和细胞因子检测。⑥结果表明：ALRM可以通过促进CD146+hAD-PCs增殖和细胞周期转化进而促进脐血CD34+造血干/祖细胞的体外扩增，在造血干细胞移植研究方面具有重要价值。
https://orcid.org/0000-0002-6768-5273 (郑波) 



中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 黑枸杞花青素, 人脂肪源性血管外膜细胞, 脐血, 造血干/祖细胞, 共培养, 体外扩增

Abstract: BACKGROUND: Anthocyanin is one of the most important active components in Lycium ruthenicum Murr, which has antioxidant and immunomodulatory effects. CD146+ human adipose-derived pericytes/perivascular cells (CD146+hAD-PCs) are the progenitors of bone marrow mesenchymal stem cells, which can promote the proliferation and differentiation of hematopoietic stem/progenitor cells in vitro. The support effect of anthocyanin in combination with CD146+hAD-PCs on umbilical cord blood hematopoietic stem/progenitor cells remains to be studied.
OBJECTIVE: To investigate the supporting effect of anthocyanins in Lycium ruthenicum Murr (ALRM) combined with CD146+hAD-PCs on umbilical cord blood CD34+hematopoietic stem/progenitor cells (UCB CD34+HSPCs) in vitro.
METHODS: The CCK-8 assay was used to detect the effect of different concentrations (0, 200, 400, 600, 800, 1 000 mg/L) of ALRM on the proliferation of CD146+hAD-PCs. Flow cytometry was used to detect the effect of ALRM on the cell cycle of CD146+hAD-PCs. The co-culture experiments were divided into blank group, ALRM group, CD146+hAD-PCs group, and ALRM+CD146+hAD-PCs group to analyze the in vitro supporting effect of ALRM combined with CD146+hAD-PCs on UCB CD34+HSPCs. The number of expanded cells and the number of colony-forming units were compared at 1, 2, and 4 weeks of co-culture. The immunophenotype of cells was detected by flow cytometry. The level of cytokines was detected by enzyme-linked immunosorbent assay.  
RESULTS AND CONCLUSION: (1) The cell viability of CD146+hAD-PCs was highest at an ALRM concentration of 200 mg/L, the proportion of G0/G1 phase cells decreased and the proportion of S and G2/M phase cells increased in CD146+hAD-PCs (P < 0.01). (2) The change in number of UCB CD34+HSPCs cells in the ALRM+CD146+hAD-PCs group was higher than that in the ALRM group at 1, 2, and 4 weeks of co-culture (all P < 0.05), and higher than that in CD146+hAD-PCs group at 2 and 4 weeks of co-culture (all P < 0.05). The number of cells in the ALRM group and blank group decreased gradually with the extension of co-culture time. (3) Colony forming capacity and immunophenotype analysis: The number of colony-forming units in the ALRM+CD146+hAD-PCs group was higher than that in the CD146+hAD-PCs group and ALRM group at 1 and 2 weeks of co-culture (P < 0.05). The proportion of CD45+ and CD34+CD33-cells in the ALRM+CD146+hAD-PCs group was higher than that in the CD146+hAD-PCs group at 1 and 2 weeks of co-culture (all P < 0.01). (4) Changes in cytokines: Interleukin-2 level in the ALRM+CD146+hAD-PCs group was higher than that in the ALRM and CD146+hAD-PCs groups (P < 0.05). The interleukin-3 content of the ALRM+CD146+hAD-PCs group was higher than that of the CD146+hAD-PCs group at 2 and 4 weeks (P < 0.05). The expression level of granulocyte colony-stimulating factor in the ALRM+CD146+hAD-PCs group was higher than that in the CD146+hAD-PCs group at 1 week, and higher than that in the ALRM group and CD146+hAD-PCs group at 2 weeks (P < 0.01). Interferon-γ content in the ALRM group and ALRM+CD146+hAD-PCs group was lower than that in the CD146+hAD-PCs group at 1, 2, and 4 weeks of co-culture (P < 0.01). (5) Due to the absence of stromal cells in the blank group, UCB CD34+HSPCs could not be counted after 1 week of co-culture and were not subjected to immunophenotyping, colony analysis, or cytokine assays. (6) In summary, ALRM can promote the expansion of UCB CD34+HSPCs in vitro by promoting CD146+hAD-PCs proliferation and cell cycle transformation, which is of great value in hematopoietic stem cell transplantation.

Key words: anthocyanins in Lycium ruthenicum Murr, human adipose-derived pericytes/perivascular cell, umbilical cord blood, hematopoietic stem/progenitor cell, co-culture, expansion in vitro

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