补肾化瘀生新方优化缺血缺氧微环境对间充质干细胞旁分泌效应及分化率的影响  

Effect of Bushen Huayu Shengxin Decoction(补肾化瘀生新方)on Paracrine Effect and Differentiation Rate of Mesenchymal Stem Cells by Optimizing Ischemic and Hypoxic Microenvironment

作　　者：张金生[1] 张宝霞[2] 刘雪曼 ZHANG Jinsheng;ZHANG Baoxia;LIU Xueman(Henan University of Chinese Medicine,Zhengzhou 450046,Henan,China;The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,Henan,China)

机构地区：[1]河南中医药大学,河南郑州450046 [2]河南中医药大学第一附属医院,河南郑州450000

出　　处：《中华中医药学刊》2021年第9期9-16,I0013,共9页Chinese Archives of Traditional Chinese Medicine

基　　金：河南省重点科技攻关项目(192102310158);河南省高校科技创新团队项目(17IRTSTHN024)。

摘　　要：目的观察补肾化瘀生新方优化缺血缺氧微环境对间充质干细胞(mesenchymal stem cells, MSCs)旁分泌效应调控作用,探讨其对MSCs增值分化能力的影响。方法采用无血清的1640培养基和低氧细胞工作站培养24 h建立缺血缺氧微环境细胞体外模型。随机分为:正常组:以完全培养基培养(L-DMEM+10%FBS);对照组:加入生理盐水血清培养基,放入低氧细胞工作站培养24 h;模型组:以完全培养基培养(L-DMEM+10%FBS),低氧细胞工作站培养24 h;治疗组:加入含有补肾化瘀生新方含药血清培养基,低氧细胞工作站培养24 h。采用液相色谱-串联质谱技术分析鉴定补肾化瘀生新方大鼠体内吸收入血成分。采用流式细胞仪检测MSCs细胞周期、ELISA法检测VEGF、bFGF、SDF-1、IL-10和TNF-α含量变化;免疫细胞化学染色检测NF-κBp65的细胞核转位;实时荧光PCR技术检测microRNA-124a-mRNA表达情况;Western blot检测microRNA-124a蛋白表达;免疫荧光技术检测MSCs向内皮细胞、神经元、神经胶质分化情况。结果与正常组比较,模型组S期细胞数增多,G0/G1期明显降低(P<0.05),与模型组比较,治疗组、对照组S期细胞数减少,G0/G1期比例逐渐升高(P<0.05),与对照组相比,治疗组S期细胞数减少,G0/G1期比例逐渐升高(P<0.05)。与正常组比较,模型组VEGF、bFGF、SDF-1、IL-10降低明显(P<0.05),与模型组比较,治疗组VEGF、bFGF、SDF-1、IL-10增多明显(P<0.05),与治疗组相比,对照组VEGF、bFGF、SDF-1、IL-10减少(P<0.05)。随缺血缺氧时间延长VEGF、bFGF、SDF-1、IL-10呈减少动态变化。与正常组比较,模型组NF-κBp65、TNF-α、microRNA-124a-mRNA和蛋白表达增多明显(P<0.05),与模型组比较,治疗组NF-κBp65、TNF-α、microRNA-124a-mRNA和蛋白表达降低明显(P<0.05),与治疗组相比,对照组NF-κBp65、TNF-α、microRNA-124a-mRNA和蛋白表达增多(P<0.05)。随缺血缺氧时间延长NF-κBp65、TNF-α、microRNA-124a-mRNA和蛋白表达呈升高�Objective To observe the regulatory effect of Bushen Huayu Shengxin Decoction(补肾化瘀生新方) on paracrine effect of mesenchymal stem cells optimized by ischemic and hypoxic microenvironment and explore its effect on the proliferation and differentiation ability of MSCs. Methods The in vitro cell model of ischemic and hypoxic microenvironment was established by culturing in serum-free 1640 medium and hypoxia cell workstation for 24 hours. The cells were randomly divided into control group(adding normal saline serum medium), model group[adding complete medium(L-DMEM+10%FBS)] and treatment group(adding serum containing Bushen Huayu Shengxin Decoction). All groups above were cultured in hypoxia cell workstation for 24 hours. While the normal group[adding complete medium(L-DMEM+10%FBS)] was cultured normally. The components of Bushen Huayu Shengxin Decoction absorbed into blood in rats were analyzed and identified by liquid chromatography-tandem mass spectrometry. The cell cycle of MSCs was detected by flow cytometry, and the content changes of VEGF, bFGF, SDF-1, IL-10, and TNF-α were detected by ELISA. The nuclear translocation of NF-κBp65 was detected by immunocytochemical staining. The expression of microRNA-124 a-mRNA was detected by real-time fluorescence PCR and the expression of microRNA-124 a protein was detected by Western Blot and the differentiation of MSCs into endothelial cells, neurons and glia were detected by immunofluorescence technique. Results Compared with that of the normal group, the number of cells in S phase was increased and the number of cells in G0/G1 phase was decreased significantly in the model group(P<0.05). Compared with that of the model group, the number of cells in S phase was decreased and the ratio of G0/G1 phase was gradually increased in the treatment group and control group(P<0.05). Compared with that of the control group, the number of cells in S phase was decreased and the ratio of G0/G1 phase was gradually increased in the treatment group(P<0.05). Compared with thos

关 键 词：补肾化瘀生新方 间充质干细胞 旁分泌效应 增殖分化 

分 类 号：R285.5[医药卫生—中药学]