中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (24): 3858-3864.doi: 10.12307/2023.422

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

电针调节糖原合成酶激酶3β/β-catenin 信号通路对脊髓损伤大鼠室管膜区细胞 CD133蛋白表达的影响

段昭远1，吴明莉2，罗  萌1，刘承梅2，高  静2，李瑞青1，2，冯晓东1，2   

  1. 1河南中医药大学，河南省郑州市  450000；2河南中医药大学第一附属医院康复中心，河南省郑州市  450000
  • 收稿日期:2022-04-06 接受日期:2022-06-20 出版日期:2023-08-28 发布日期:2023-01-19
  • 通讯作者: 冯晓东，主任医师，博士生导师，河南中医药大学第一附属医院康复中心，河南省郑州市 450000；河南中医药大学，河南省郑州市 450000
  • 作者简介:段昭远，男，1993年生，河南省平顶山市人，汉族，河南中医药大学在读硕士，主要从事脊髓损伤康复治疗及相关研究。
  • 基金资助:
    河南省科技厅重点研发与推广专项(202102311130)，项目负责人：吴明莉；2019年度河南省中医药科学研究专项课题(2019ZY2132)，项目负责人：吴明莉；河南省高等教育学校重点科研项目(22A360011)，项目负责人：刘承梅

Effect of electroacupuncture modulation of glycogen synthase kinase 3 beta/beta-catenin signaling pathway on CD133 protein expression in rat ventricular zone cells after spinal cord injury

Duan Zhaoyuan1, Wu Mingli2, Luo Meng1, Liu Chengmei2, Gao Jing2, Li Ruiqing1, 2, Feng Xiaodong1, 2   

  1. 1Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China; 2Rehabilitation Center, the First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China
  • Received:2022-04-06 Accepted:2022-06-20 Online:2023-08-28 Published:2023-01-19
  • Contact: Feng Xiaodong, Chief physician, Doctoral supervisor, Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China; Rehabilitation Center, the First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China
  • About author:Duan Zhaoyuan, Master candidate, Henan University of Chinese Medicine, Zhengzhou 450000, Henan Province, China
  • Supported by:
    Henan Provincial Science and Technology Department Key R&D and Promotion Special Project, No. 202102311130 (to WML); 2019 Henan Provincial TCM Scientific Research Special Project, No. 2019ZY2132 (to WML); Key Scientific Research Project of Higher Education Schools in Henan Province, No. 22A360011 (to LCM)

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摘要：

文题释义：

CD133蛋白：是神经干细胞的表面标志之一，在细胞生长、发育和肿瘤生物学等方面都有作用。研究表明，CD133 阳性能普遍地代表静息状态下的成体神经干细胞 。
内源性神经干细胞：是指体内神经系统中存在的具有多分化潜能、能够分化成神经元和其他胶质细胞并能实现自我更新的一类细胞，广泛分布于哺乳动物胚胎的中枢神经系统。

背景：前期研究表明，电针可改善脊髓损伤后功能障碍，并促进脊髓损伤后大鼠内源性神经干细胞增殖，然而其具体作用机制不明确。 
目的：观察电针对脊髓损伤大鼠糖原合成酶激酶3β/β-catenin 信号通路相关因子表达和室管膜区细胞CD133蛋白表达的影响，探讨电针促进内源性干细胞增殖的作用机制。
方法：将 45 只 SD 雌性大鼠随机分为假手术组、模型组、电针组，每组 15 只，后两组以精密打击器构建脊髓损伤大鼠模型。在造模后
24 h，电针组接受电针治疗，选取督脉“百会”“脊中”穴及损伤脊髓上下节段夹脊穴，予疏密波，频率1 Hz/20 Hz，强度 1.0-1.2 mA，30 min/次，1次/d，持续 14 d。应用BBB 运动功能评分评估大鼠运动功能，Nissl 染色观察脊髓组织病理形态学改变及神经元数量，免疫荧光检测脊髓组织中室管膜区细胞 CD133、脊髓灰质和白质中 Nestin 荧光强度，实时荧光定量PCR 检测脊髓组织中 Nestin、糖原合成酶激酶3β、β-catenin mRNA 表达，Western blot 检测脊髓组织中 Nestin、糖原合成酶激酶3β、磷酸化糖原合成酶激酶3β、β-catenin、p-β-catenin 蛋白表达。

结果与结论：①与模型组比较，电针组BBB 运动功能评分术后 1，3，5 d 时无显著差异(P > 0.05)，术后7，14 d时显著提高(P < 0.05、P < 0.01)；②病理形态上，电针组脊髓组织部分神经细胞形态较完整，尼氏小体溶解较轻；与模型组比较，电针组脊髓组织神经元数量增加(P < 0.05)；③与模型组比较，电针组脊髓组织中室管膜区 CD133荧光强度升高(P < 0.001)，灰质中Nestin荧光强度均明显升高(P < 0.01)，白质中 Nestin 荧光强度显著升高(P < 0.001)；④与模型组比较，电针组Nestin mRNA表达水平显著升高(P < 0.001)，糖原合成酶激酶3β mRNA 表达水平明显降低(P < 0.01)，β-catenin mRNA 表达水平显著降低(P < 0.001)；⑤与模型组比较，电针组 Nestin、磷酸化糖原合成酶激酶3β蛋白表达水平显著升高(P < 0.001)，p-β-catenin 蛋白表达水平显著降低(P < 0.001)；⑥结果表明，电针能够促进脊髓损伤后大鼠室管膜区 CD133蛋白表达，其机制可能与调节糖原合成酶激酶3β/β-catenin 信号通路有关。

https://orcid.org/0000-0001-7414-2923(段昭远)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 电针, 脊髓损伤, 室管膜细胞, 内源性神经干细胞, 糖原合成酶激酶3β

Abstract: BACKGROUND: Preliminary studies have shown that electroacupuncture can improve functional impairment after spinal cord injury and promote the proliferation of endogenous neural stem cells in rats after spinal cord injury. However, its specific mechanism of action is unclear.
OBJECTIVE: To investigate the effect of electroacupuncture on the expression of glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling pathway-related factors and CD133 protein in ventricular canal area cells in rats with spinal cord injury, and to explore the mechanism by which electroacupuncture promotes endogenous stem cell proliferation.
METHODS: A total of 45 female Sprague-Dawley rats were randomly divided into sham-operated group, model group, and electroacupuncture group, with 15 rats in each group. Animal models of spinal cord injury were constructed in the latter two groups using a precision percussion device. The electroacupuncture group received electroacupuncture treatment at Baihui and Jizhong acupoints of the Governor’s Vessel and at Jiaji acupoints of the upper and lower segments of the injured spinal cord, with sparse and dense waves at a frequency of 1 Hz/20 Hz and an intensity of 1.0-1.2 mA, once for 30 minuntes, once a day, for 14 continuous days. The motor function of the rats was assessed using the Basso, Beattie and Bresnahan motor function score and Nissl staining was used to observe the histomorphological changes and the number of neurons in the spinal cord. Immunofluorescence was used to detect the fluorescence intensity of CD133 in ependymal cells in spinal cord tissue and Nestin in spinal cord gray matter and white matter. Real-time fluorescence quantitative PCR was used to detect Nestin, GSK-3β, and β-catenin mRNA in spinal cord tissue. Western blot assay was used to detect Nestin, GSK-3β, p-GSK-3β, β-catenin, and p-β-catenin protein expression in spinal cord tissue. 
RESULTS AND CONCLUSION: (1) Compared with the model group, the Basso, Beattie and Bresnahan scores showed no significant changes in the electroacupuncture group at 1, 3, and 5 days after surgery (P > 0.05) but were significantly increased at 7 and 14 days after surgery (P < 0.05, P < 0.01). (2) The morphology of some nerve cells in the spinal cord tissue was relatively intact in the electroacupuncture group and Nissl bodies were slightly dissolved. Compared with the model group, the number of neurons in the spinal cord tissue was significantly increased in the electroacupuncture group (P < 0.05). (3) Compared with the model group, in the electroacupuncture group, the fluorescence intensity of CD133 in the ventricular zone of spinal cord tissue was increased (P < 0.001), and the fluorescence intensity of Nestin was significantly increased in the gray matter (P < 0.01) and white matter (P < 0.001). (4) Compared with the model group, in the electroacupuncture group, the expression of Nestin mRNA was significantly higher (P < 0.001), GSK-3β mRNA expression was significantly lower (P < 0.01), and β-catenin mRNA expression was also significantly lower (P < 0.001). (5) Compared with the model group, Nestin and p-GSK-3β protein expression was significantly higher (P < 0.001) and p-β-catenin protein expression was significantly lower (P < 0.001) in the electroacupuncture group. (6) All these findings indicate that electroacupuncture can promote CD133 protein expression in the ventricular canal area of rats after spinal cord injury, and the mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway.

Key words: electroacupuncture, spinal cord injury, ependymal cell, endogenous neural stem cell, glycogen synthase kinase 3β

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