摘要
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(0,200,400,600,800,1000 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:(1)ALRM质量浓度为200 mg/L时,CD146^(+)hAD-PCs活力最高,CD146^(+)hAD-PCs的G_(0)/G_(1)期细胞比例下降,S期、G_(2)/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^(+)h AD-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。
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(0,200,400,600,800,1000 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 withCD146~+h AD-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 G_0/G_(1)phase cells decreased and the proportion of S and G_2/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~+h AD-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~+h AD-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~+h AD-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~+h AD-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 colonystimulating 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~+h AD-PCs group at 2 weeks(P<0.01).Interferon-γcontent 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.
作者
申娅媚
牛云霞
杨婷婷
马洁
胡代宏
郑波
Shen Yamei;Niu Yunxia;Yang Tingting;Ma Jie;Hu Daihong;Zheng Bo(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;Diagnosis and Treatment Engineering Technology Research Center of Nervous Disease of Ningxia,General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China;Department of Hematology,General Hospital of Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China)
出处
《中国组织工程研究》
CAS
北大核心
2025年第1期58-64,共7页
Chinese Journal of Tissue Engineering Research
基金
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。
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
anthocyanins in Lycium ruthenicum Murr
human adipose-derived pericytes/perivascular cell
umbilical cord blood
hematopoietic stem/progenitor cell
co-culture
expansion in vitro
