实验动物与比较医学 ›› 2024, Vol. 44 ›› Issue (5): 531-542.DOI: 10.12300/j.issn.1674-5817.2024.031

• 人类疾病动物模型 • 上一篇    下一篇

经血干细胞移植联合运动训练促进大鼠脊髓损伤康复的转录组学分析

戚龙菊1(), 陈世园1,3, 廖泽华1,3, 石袁虎1,3, 孙郁雨1, 王庆华2()()   

  1. 1.南通大学附属南通第三医院, 南通市第三人民医院, 南通 226000
    2.南通大学实验动物中心, 南通 226001
    3.南通大学医学院, 南通 226001
  • 收稿日期:2024-02-27 修回日期:2024-08-06 出版日期:2024-11-06 发布日期:2024-10-25
  • 通讯作者: 王庆华(1978—),男,博士,讲师,研究方向为脊髓损伤后功能康复。E-mail: wangqh@ntu.edu.cn。ORCID: 0000-0003-3661-6408
  • 作者简介:戚龙菊(1985—),女,硕士,副主任护师,研究方向为脊髓损伤后功能康复。E-mail: qilongjunt@163.com
  • 基金资助:
    南通市科技计划项目“GelMA水凝胶负载MenSCs-Exo调控miR-421 / MAGED1 /PI3K-Akt分子轴改善脊髓损伤后功能恢复的机制研究”(JC2023040);“MenSCs外泌体调控miR-421/MAGED1/PPARγ轴改善脊髓损伤后轴突再生机制及其临床诊疗技术攻关”(MS22022012);南通市卫生健康委员会科研课题面上项目“有氧运动调控GDNF减轻脊髓损伤后疼痛的机制及临床应用研究”(MS2022062)

Transcriptomic Analysis of Menstrual Blood-Derived Stem Cells Transplantation Combined with Exercise Training in Promoting Spinal Cord Injury Recovery in Rats

QI Longju1(), CHEN Shiyuan1,3, LIAO Zehua1,3, SHI Yuanhu1,3, SUN Yuyu1, WANG Qinghua2()()   

  1. 1.Affiliated Nantong Third Hospital of Nantong University, Nantong Third People's Hospital, Nantong 226000, China
    2.Laboratory Animal Center of Nantong University, Nantong 226001, China
    3.Medical School of Nantong University, Nantong 226001, China
  • Received:2024-02-27 Revised:2024-08-06 Published:2024-10-25 Online:2024-11-06
  • Contact: WANG Qinghua (ORCID: 0000-0003-3661-6408), E-mail: wangqh@ntu.edu.cn

摘要:

目的 通过转录组测序分析探讨经血干细胞(menstrual blood-derived stem cells,MenSCs)移植联合运动训练治疗脊髓损伤(spinal cord injury,SCI)大鼠的潜在干预靶点和分子机制。 方法 选用SPF级2月龄雌性SD大鼠,采用第十胸椎(T10)处半切方式构建SCI模型后,分为SCI后MenSCs移植联合运动训练组[简称细胞与跑步机训练(cell and treadmill training,CTMT)组]和SCI组(作为对照),每组12只大鼠。其中,CTMT组大鼠在建模后1周于损伤局部显微注射MenSCs 1×105个,随后进行为期2周的减重有氧运动训练。选取损伤处的脊髓组织进行转录组测序分析,获得SCI组和CTMT组大鼠的脊髓组织中mRNA表达数据,进行基因表达差异分析、GO功能富集分析、KEGG通路富集分析和蛋白质互作网络分析。同时,采用BBB评分评估两组大鼠的运动功能康复情况,苏木精-伊红(hematoxylin and eosin,HE)染色评估两组大鼠损伤局部的组织病理学改善程度,采用实时荧光定量PCR法和蛋白质印迹法对差异基因表达进行验证。 结果 转录组测序分析差异基因的表达数据显示,与SCI组相比,CTMT组有247个上调基因及174个下调基因,其中BdnfHmox1、Sd4、Mmp3和Cd163等基因显著上调[|log2(FoldChange)|≥0.66,P<0.05]。KEGG通路富集分析与GO功能富集分析提示,这些差异基因主要参与了生长发育、代谢反应及免疫炎症过程,例如轴突生长、电子传递链等。其中,Bdnf基因富集在PI3K-Akt信号通路。BBB评分显示,MenSCs移植联合运动训练显著提高SCI大鼠的运动能力。HE染色提示治疗组大鼠的损伤局部病理变化程度显著减轻。实时荧光定量PCR法和蛋白质印迹法证明,CTMT组脊髓组织中脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)mRNA和蛋白表达水平均显著高于SCI组(P<0.001)。 结论 MenSCs移植联合运动训练治疗可能通过上调BDNF表达促进SCI大鼠运动功能的恢复,这为SCI的临床康复治疗提供了一个新思路。

关键词: 脊髓损伤, 转录组测序, 经血干细胞, 运动训练, 脑源性神经生长因子, 大鼠

Abstract:

Objective To explore the potential therapeutic targets and molecular mechanisms of menstrual blood-derived stem cells (MenSCs) transplantation combined with exercise training in promoting recovery in rats with spinal cord injury (SCI) through transcriptome sequencing analysis. Methods Female SD rats aged two months were selected and a SCI model was established by a hemisection at the tenth thoracic vertebra (T10). The rats were then divided into two groups: the Cell and Treadmill Training (CTMT) group, which received MenSCs transplantation and treadmill training after SCI, and the SCI group (control), with 12 rats in each group. One week after modeling, the CTMT group received a microinjection of 1×105 MenSCs at the injury site, followed by two weeks of weight-supported aerobic exercise training. Spinal cord tissue from the injury site was selected for transcriptome sequencing, and mRNA expression data from both the SCI and CTMT groups were analyzed. Differential gene expression, GO (Gene Ontology) functional enrichment, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment, and protein-protein interaction (PPI) network analyses were performed. Motor function recovery was assessed using the Basso, Beattie, and Bresnahan (BBB) score, while histopathological changes at the injury site were evaluated through hematoxylin-eosin (HE) staining. Real-time fluorescent quantitative PCR and Western blotting were used to verify the expression of differentially expressed genes. Results Transcriptome sequencing analysis showed 247 upregulated genes and 174 downregulated genes in the CTMT group compared to the SCI group. Notably, genes such as Bdnf, Hmox1, Sd4, Mmp3, and Cd163 were significantly upregulated [|log2(FoldChange)|≥0.66, P<0.05]. KEGG pathway enrichment analysis and GO functional enrichment analysis indicated that these differentially expressed genes were mainly involved in growth and development, metabolic reactions, and immune-inflammatory processes, such as axon growth and the electron transport chain. The Bdnf gene was notably enriched in the PI3K-Akt signaling pathway. The BBB score showed that MenSCs transplantation combined with exercise training significantly improved the motor function of SCI rats. HE staining revealed that pathological changes at the injury site were significantly reduced in the treatment group. Furthermore, real-time quantitative PCR and Western blotting confirmed that brain-derived neurotrophic factor (BDNF) mRNA and protein expression levels in the CTMT group were significantly higher than those in the SCI group (P<0.001). Conclusion The combined exercise training with MenSCs effectively promotes the recovery of motor function in SCI rats by upregulating BDNF expression, providing a novel strategy for SCI treatment.

Key words: Spinal cord injury, Transcriptome sequencing, Menstrual blood-derived stem cells, Exercise training, Brain-derived neurotrophic factor, Rats

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