解剖学报 ›› 2024, Vol. 55 ›› Issue (5): 556-564.doi: 10.16098/j.issn.0529-1356.2024.05.006

• 细胞和分子生物学 • 上一篇    下一篇

当归多糖促进骨髓移植重建受体小鼠造血功能

杨婷1  廖奎2  黄彩虹1  魏晗1  王程1  杜坤航1 汪子铃1  王璐1  王亚平1*
  

  1. 1.重庆医科大学组织学与胚胎学教研室,干细胞与组织工程研究室;2.第一附属医院肿瘤科,重庆  400016
  • 收稿日期:2024-05-28 修回日期:2024-06-09 出版日期:2024-10-06 发布日期:2024-10-06
  • 通讯作者: 王亚平 E-mail:ypwangcq@aliyun.com
  • 基金资助:
    当归多糖调控Keap1-Nrf2-ARE抗氧化通路延缓造血干细胞 衰老的机制研究;当归多糖通过调控网状基质细胞分泌IL-7修复化疗后Pro-B淋巴细胞的机制研究

Angelica Sinensis polysaccharide promoting hematopoietic reconstruction in receptor mice after bone marrow transplantation

YANG  Ting1  LIAO  Kui2  HUANG  Cai-hong1  WEI  Han1  WANG  Cheng1  DU  Kun-hang1  WANG  Zi-ling1  WANG  Lu1  WANG  Ya-ping1* #br#

#br#
  

  1. 1.Department of Histology and Embryology, Laboratory of Stem Cells and Tissue Engineering,Chongqing Medical University;  2.Department of Oncology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016,China
  • Received:2024-05-28 Revised:2024-06-09 Online:2024-10-06 Published:2024-10-06
  • Contact: WANG Ya-ping E-mail:ypwangcq@aliyun.com

摘要:

目的   探讨当归多糖(ASP)调控骨髓基质细胞(BMSCs)促进供体骨髓移植(BMT)重建受体小鼠造血功能的机制。 方法   分离纯化8~10周龄雄性C57BL/6J小鼠骨髓单个核细胞(BMMNCs),移植给同龄同种雌性受体小鼠,第9天取受体鼠BMMNCs,再次移植给雌性受体小鼠。移植受体鼠分为对照组:假照射;辐射组:8.0 Gy X射线全身一次性照射;骨髓移植组:照射同辐射组,经尾静脉移植雄性供体BMMNCs(5×106个细胞);骨髓移植ASP组:同骨髓移植组,从移植的第1天起连续腹腔注射ASP[100 mg/(kg·d)×9]。模型构建期间记录小鼠体重与生存变化,模型构建结束后采集受体小鼠BMMNCs检测Y染色体性别决定区(SRY)基因,测定外周血血常规指标,计算股骨BMMNCs数,观察骨髓组织病理学;分离培养受体鼠BMSCs,观察细胞贴壁生长,5-乙炔基-2’-脱氧尿苷(EdU)法检测BMSCs增殖;分析细胞内活性氧(ROS)水平、超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量;检测BMSCs培养上清液中粒细胞-巨噬细胞集落刺激因子(GM-CSF)、干细胞因子(SCF)和胰岛素样生长因子1(IGF-1)水平,检测BMMNCs与各组BMSCs共培养48h,形成造血祖细胞混合集落(CFU-Mix)数量;Real-time PCR分析受体BMMNCs的Notch信号通路相关基因(Notch1、Jagged1、Hes1)表达。 结果   单纯辐射组小鼠全部死亡,骨髓移植ASP组受体鼠体重下降不明显;在受体雌鼠BMMNCs中检测到SRY基因;骨髓移植ASP组受体鼠外周血血常规和BMMNCs总数下降幅度不显著,骨髓组织病理学损伤减轻;ASP能促进BMSCs增殖,降低BMSCs中 ROS、MDA含量,提高SOD活性;促进BMSCs分泌SCF、GM-CSF及IGF-1,提高BMMNCs与受体BMSCs共培养后的CFU-Mix产率;提高受体小鼠BMMNCs中Notch1、Jagged1、Hes1 mRNA表达。 结论   ASP促进受体小鼠造血功能重建机制与减轻造血微环境氧化应激损伤,提高BMSCs分泌造血生长因子,调控Notch信号通路有关。 

关键词: 当归多糖|X射线辐射|骨髓移植|造血重建|骨髓微环境|免疫荧光|实时定量聚合酶链反应|小鼠 

Abstract:

Objective   To explore the mechanism of Angelica Sinensis polysaccharide (ASP) promoting donor bone marrow transplantation (BMT) to reconstruct hematopoietic function of receptor mice by regulating bone marrow stromalcells (BMSCs).  Methods   Bone marrow mononuclear cells (BMMNCs) of male C57BL/6J mice aged 8-10 weeks were separated, purified and transplanted into female receptor mice of the same age. On the ninth day, receptor mice BMMNCs were separated, purified and transplanted again into female receptor mice. The transplanted receptor mice were divided into control group:  sham irradiation; Irradiation(IR) group: a whole-body irradiation with a total dose of 8.0 Gy X-ray; BMT group: the receptor mice treated in the same way as the IR group and transplanted BMMNCs (5×106 cells) from male donor via the tail vein; BMT+ASP group: the receptor mice treated in the same way as the BMT group, and injected ASP [100 mg/(kg·d)×9] by intraperitoneal route from the first day of transplantation. Changes in body weight and survival rate of mice were recorded during modeling, receptor mice BMMNCs were collected to detect sexdetermining region of Y(SRY) gene after building model, peripheral blood indexes, the number of BMMNCs in femur and histopathology of bone marrow were detected; BMSCs in receptor mice was separated and purified, BMSCs adhesion ability was observed, proliferation ability was detected by 5-ethynyl-2-deoxyuridine(EdU);The level of reactive oxygen species (ROS), the activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in BMSCs were detected; The levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor(SCF), insulinlike growth factor 1(IGF-1) in culture supernatant of BMSCs were determined, CFU-Mix was counted after BMMNCs co-cultured with receptor BMSCs in each group for 48 hours;The expression of Notch signaling pathway related genes (Notch1, Jagged1, Hes1) in BMMNCs were measured by Real-time PCR.    Results   All mice in IR group were died,the body weight loss in BMT+ASP group was not obvious. The SRY gene was detected in the receptor female mice BMMNCs. Peripheral blood indexes and the number of BMMNCs were not significantly decreased in BMT+ASP group receptor mice,and bone marrow histopathological injury was reduced. ASP promoted the proliferation of BMSCs, decreased the contents of ROS and MDA, and increased the activity of SOD in BMSCs. ASP promoted the secretion of SCF, GM-CSF and IGF-1 in BMSCs, and increased CFU-Mix yield of BMMNCs co-cultured with receptor BMSCs. ASP increased the expression of Notch1, Jagged1 and Hes1 mRNA in BMMNCs.  Conclusion   The mechanism of ASP promoting receptor hematopoietic function reconstruction is related to reducing the oxidative stress damage of hematopoietic microenvironment, improving the secretion of hematopoietic growth factors in BMSCs, and regulating Notch signaling pathway. 

Key words: Angelica Sinensis polysaccharide|X-ray radiation, Bone marrow transplantation|Hematopoietic reconstruction|Bone marrow microenvironment|Immunofluorescence|Real-time PCR|Mouse

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