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

doi:10.12307/2024.738

Abstract

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

CLC Number:

Shen Yamei, Niu Yunxia, Yang Tingting, Ma Jie, Hu Daihong, Zheng Bo. Anthocyanins from Lycium ruthenicum Murr combined with human adipose-derived pericytes/perivascular cells support proliferation of umbilical cord blood hematopoietic stem/progenitor cells[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(1): 58-64.

Figures/Tables 1

2.1 ALRM对CD146+hAD-PCs增殖的影响 CCK-8法检测结果显示，与0 mg/L相比，当ALRM质量浓度为200 mg/L时，CD146+hAD-PCs活力最高(P < 0.001)，且随着ALRM质量浓度的升高，CD146+hAD-PCs活力逐渐下降。故选择200 mg/L ALRM作为促进CD146+hAD-PCs增殖的最佳质量浓度，见图1。 2.2 ALRM对CD146+hAD-PCs细胞周期的影响流式细胞术检测结果显示，用含有200 mg/L ALRM的高糖型DMEM培养基处理后CD146+hAD-PCs(实验组)G0/G1期、S期、G2/M期细胞比例分别为(71.52±1.49)%，(26.25±1.76)%，(8.17±1.42)%，对照组G0/G1期、S期、G2/M期细胞比例分别为(86.15±1.22)%，(10.18±1.72)%，(2.73±1.66)%，两组比较差异有显著性意义(P < 0.01，P < 0.001)，见图2。 2.3 各培养体系UCB CD34+HSPCs镜下生长状态接种当天(0周)，在倒置显微镜下观察可见UCB CD34+HSPCs呈悬浮状态，体积小而透亮，圆形，分布均匀，CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组可见梭形基质细胞(CD146+hAD-PCs)，贴壁生长，见图3。共培养1周时，CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组出现UCB CD34+HSPCs沿基质细胞聚集的现象，体积较0周时略有增大，折光性良好，空白组和ALRM组因无基质细胞，UCB CD34+HSPCs体积缩小，密度降低，折光性减弱，见图3。共培养2周时，CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组的UCB CD34+HSPCs数量明显增加，密度增大，沿基质细胞分布，圆形透亮具有折光性，空白组和ALRM组UCB CD34+HSPCs数量明显减少，空白组仅可见散在细胞，折光性差，见图3。共培养4周时，CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组的UCB CD34+HSPCs沿底部基质细胞周围分布，密度减低，大部分细胞折光性变差，ALRM组散在分布少量的UCB CD34+HSPCs，已无折光性，空白组UCB CD34+HSPCs全部死亡，见图3。 2.4 各培养体系UCB CD34+HSPCs细胞数量变化分析共培养1周时，各组的UCB CD34+HSPCs数量均有下降，ALRM+CD146+hAD-PCs组与CD146+hAD-PCs组细胞数量差异无显著性意义(P > 0.05)，ALRM+CD146+hAD-PCs组比ALRM组细胞数量多(P < 0.01)，CD146+hAD-PCs组比空白组细胞数量多(P < 0.01)，空白组、ALRM组、CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组的细胞数量分别为(0.33±0.05)×104，(0.65±0.20)×104，(1.66±0.03)×104，(2.25±0.25)×104个/孔。共培养2周时，空白组、ALRM组、CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组的细胞数量分别为(0.17±0.02)×104，(0.40±0.15)×104，(2.13±0.13)×104，(3.38±0.10)×104个/孔，CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组细胞数量增加，空白组、ALRM组细胞数量持续减少，与CD146+hAD-PCs组相比，ALRM+CD146+hAD-PCs组细胞数量显著增加(P < 0.001)，与ALRM组相比，ALRM+CD146+hAD-PCs组细胞数量显著增加(P < 0.001)，CD146+hAD-PCs组细胞数量也显著高于空白组(P < 0.001)。培养4周时，空白组已无细胞，ALRM、CD146+hAD-PCs组、ALRM+CD146+hAD-PCs组细胞数量较2周时增加，分别为(0.89±0.51)×104，(2.34±0.14)×104，(4.31±0.01)×104个/孔；此时，ALRM+CD146+hAD-PCs组细胞数量仍显著高于CD146+hAD-PCs组(P < 0.001)、ALRM组(P < 0.001)，见图4。 2.5 各培养体系UCB CD34+HSPCs集落形成能力空白组UCB CD34+HSPCs细胞数量持续减少，在共培养的各时间点均无集落形成。ALRM组UCB CD34+HSPCs在共培养1，2，4周的集落形成单位数量分别为(12.50±4.50)，(8.25±1.25)，(1.00±0.10)个，CD146+hAD-PCs组各时间点集落形成单位数量为(36.00±5.00)，(15.50±7.50)，(4.75±0.75)个，ALRM+CD146+hAD-PCs组各时间点的集落形成单位数量为(65.50±13.50)，(40.00±5.00)，(10.50±1.50)个，见图5。共培养1，2周，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)。 2.6 共培养后UCB CD34+HSPCs表型变化共培养1，2，4周时，与CD146+hAD-PCs组相比，ALRM+CD146+hAD-PCs组CD45+、CD34+CD33-细胞比例升高，差异有显著性意义(P < 0.01，P < 0.01)，见图6。空白组和ALRM组无基质细胞作为滋养层，UCB CD34+HSPCs细胞计数在第1，2周显著减少，4周已无存活，无法进行流式细胞免疫表型分析。 2.7 各组共培养体系上清液细胞因子水平变化 ELISA法检测各组共培养体系上清液中细胞因子分泌水平，见图7，ALRM+CD146+hAD-PCs组在4周时白细胞介素2水平高于ALRM组、CD146+hAD-PCs组(P < 0.05)。ALRM+CD146+hAD-PCs组在2，4周时白细胞介素3水平高于CD146+hAD-PCs组(P < 0.05，P < 0.05)。ALRM+CD146+hAD-PCs组在1周时粒细胞集落刺激因子水平高于CD146+hAD-PCs组(P < 0.001)，2周时粒细胞集落刺激因子水平高于ALRM组、CD146+hAD-PCs组(P < 0.01，P < 0.01)。CD146+hAD-PCs组在1，2，4周时的干扰素γ水平高于ALRM组(P < 0.05，P < 0.01，P < 0.001)；ALRM+CD146+hAD-PCs组在1，2，4周时干扰素γ水平低于CD146+hAD-PCs组(P < 0.05，P < 0.01，P < 0.001)；ALRM组与CD146+hAD-PCs组干扰素γ水平在共培养的各个时间无统计学差异。空白组UCB CD34+HSPCs数量持续减少，第2周已无细胞存活，未进行上清液细胞因子检测。"

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