杜仲提取物增强骨髓基质细胞移植对创伤性脑损伤治疗效能相关性研究
The relationship of Eucommia ulmoides promoting the therapeutic efficacy of bone marrow stromal cells transplantation for traumatic brain injury
刘欣 1刘亮 1张旭2
作者信息
- 1. 北部战区总医院 神经内科,辽宁 沈阳 110016
- 2. 北部战区总医院 心血管外科,辽宁 沈阳 110016
- 折叠
摘要
目的 探讨杜仲(EU)提取物对骨髓基质细胞(BMSCs)生物学特性及其移植治疗创伤性脑损伤(TBI)效能的影响.方法 体外实验:将C57BL/6 小鼠来源BMSCs分为EU组和Control组(每组各8 只),分别给予EU提取物(100 μg/ml)和等量溶剂.通过相差显微镜观察和CCK-8 试验检测细胞形态增殖情况.通过免疫荧光(IF)染色和定量逆转录聚合酶链反应(qRT-PCR)检测细胞神经分化相关基因[Oct4、神经原纤维轻链(Nefl)、神经分化因子(Neurod1)、神经生长因子(Ngf)]表达情况.体内实验:将32 只3 月龄雄性C57BL/6 小鼠随机分为Sham surgery组、TBI组、TBI +BMSCs组和TBI +EU_BMSCs组(每组各8 只).对TBI组、TBI +BMSCs组和TBI +EU_BMSCs组进行TBI手术,术后 7 d,TBI组给予 100 μl PBS,TBI + BM-SCs组经眶内静脉注射无特殊处理的GFP转基因小鼠来源BMSCs,TBI +EU_BMSCs组经眶内静脉注射等量EU提取物预处理14d的GFP转基因小鼠来源BMSCs.Sham surgery组接受除TBI手术及细胞注射移植以外的其他操作.通过转棒式疲劳仪和平衡木行走试验检测运动功能.通过IF染色进行组织病理学检测.结果 体外实验:EU提取物处理14d后,相差显微镜图像分析和CCK-8 试验结果显示,EU组增殖活性高于Control组(P<0.001);qRT-PCR检测显示,EU组Neurod1 和Ngf mRNA表达量高于Control组(P<0.001).EU提取物处理70d后,EU组细胞呈神经元样形态;IF染色显示,EU组较高比例细胞表达Nefl和MAP2,而Control组未检出(P<0.001);qRT-PCR分析显示,EU组Oct4 mRNA表达量低于Control组,而Ne-fl、Neurod1 和Ngf转录水平高于Control组(P<0.001).体内实验:转棒式疲劳仪和平衡木行走试验显示,TBI + BMSCs组小鼠运动功能恢复明显优于TBI组(P<0.001),TBI +EU_BMSCs组小鼠运动功能改善程度较TBI +BMSCs组进一步提高(P<0.001).脑组织IF染色显示,TBI +BMSCs组小鼠损伤灶内MAP2 表达水平高于TBI组(P<0.001),TBI + EU_BMSCs组小鼠MAP2 表达量进一步恢复(P<0.001).TBI +EU_BMSCs组小鼠损伤灶内GFP +移植细胞含量及分化为MAP2 +细胞的GFP +移植细胞比例均显著高于TBI +BMSCs组(P<0.001).结论 EU提取物处理可促进BMSCs向神经元分化,并显著提高BMSCs移植治疗TBI的效能.
Abstract
Objective To investigate the effects of Eucommia ulmoides(EU)on the biological features of bone marrow stromal cells(BMSCs)and the therapeutic efficacy of grafted BMSCs for traumatic brain injury.Methods In vitro,the C57BL/6 mice-derived BMSCs were divided into EU and Control groups(8 in each group),which were treated with EU extract(100 ng/ml)and the dissol-vent of the equal volume respectively.Phase contrast microscopy and CCK-8 assay were used to detect cell morphology and prolifera-tion.Immunofluorescence(IF)staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR)were used to de-tect the expression of genes related to neural differentiation[Oct4,neurofibrilar light chain(Nefl),neuro differentiation factor(Neurod1),and nerve growth factor(Ngf)].In vivo,thirty-two 3-month old male C57BL/6 mice were divided randomly into Sham surgery,TBI,TBI +BMSCs and TBI +EU_BMSCs groups(8 in each group).The TBI group,TBI +BMSCs group and TBI +EU_BM-SCs group were treated with 100 μl PBS 7 days after operation,and the TBI +BMSCs group was injected with GFP transgenic murine BMSCs without special treatment via intraorbital vein.GFP transgenic mouse derived BMSCs in TBI +EU_BMSCs group were pretrea-ted with the same amount of EU extract by intraorbital vein for 14 days.Mice in the Sham surgery group underwent all procedures ex-cept TBI surgery and cell transplantation.The locomotor of mice was monitored by Rotarod and Beam walking tests.The histopathologi-cal analyses were performed by IF staining.Results In vitro experiments,after 14 days of EU,phase contrast microscope image analy-sis and CCK-8 test showed that the proliferation activity of EU group was higher than that of Control group(P<0.001).The expres-sion levels of Neurod1 and Ngf mRNA in EU group were higher than those in Control group by qRT-PCR(P<0.001).After treated with EU extract for 70 days,the cells in EU group showed neuron-like morphology.IF staining showed that Nefl and MAP2 were ex-pressed in a higher proportion of cells in EU group,but not in Control group(P<0.001).qRT-PCR analysis showed that the expres-sion of Oct4 mRNA in EU group was lower than that in Control group,while the transcription levels of Nefl,Neurod1 and Ngf were higher than that in Control group(P<0.001).In vivo experiment,the rotating rod fatigue meter and balance beam walking tests showed that the recovery of motor function in TBI +BMSCs group was significantly better than that in TBI group(P<0.001),and the improvement of motor function in TBI +EU_BMSCs group was further improved than that in TBI +BMSCs group(P<0.001).IF stai-ning of brain tissue showed that the expression level of MAP2 in the lesion of TBI +BMSCs group was higher than that of TBI group(P<0.001),and the expression level of MAP2 in TBI +EU_BMSCs group was further recovered(P<0.001).The content of GFP + transplanted cells and the proportion of GFP + transplanted cells differentiated into MAP2 + cells in TBI +EU_BMSCs group were sig-nificantly higher than those in TBI +BMSCs group(P<0.001).Conclusion EU extract promoted the differentiation of BMSCs into neurons and enhanced the therapeutic efficacy of grafted BMSCs for TBI.
关键词
杜仲/骨髓基质细胞/分化/移植/创伤性脑损伤Key words
Eucommia ulmoides/Bone marrow stromal cells/Differentiation/Transplantation/Traumatic brain injury引用本文复制引用
基金项目
辽宁省博士科研启动基金(2019-BS-269)
出版年
2024
NETL
NSTL
维普
万方数据