中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (19): 2970-2977.doi: 10.12307/2022.373

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

睫状神经营养因子复合毛喉素及3-异丁基-1-甲基黄嘌呤通过环磷酸腺苷信号通路诱导肌源性干细胞分化为许旺细胞表型

龚  超1，智晓东1，2，3，张玉强1，2，3，王晨亮1，王  康1，王  伟1，2，3   

  1. 1锦州医科大学附属第一医院骨科，辽宁省锦州市   121000；2锦州医科大学骨外科学研究所，辽宁省锦州市   121000；3辽宁省医学组织工程重点实验室，辽宁省锦州市   121000
  • 收稿日期:2021-01-08 修回日期:2021-02-05 接受日期:2021-07-19 出版日期:2022-07-08 发布日期:2021-12-28
  • 通讯作者: 王伟，博士，主任医师，教授，锦州医科大学附属第一医院骨科，辽宁省锦州市 121000；锦州医科大学骨外科学研究所，辽宁省锦州市 121000；辽宁省医学组织工程重点实验室，辽宁省锦州市 121000
  • 作者简介:龚超，男，1993年生，湖北省十堰市人，汉族，锦州医科大学在读硕士，主要从事周围神经损伤修复重建与再生相关研究。
  • 基金资助:
    锦州医科大学校企合作基金项目(2020002)，项目负责人：王伟；辽宁省科学技术厅科学技术基金面上项目(20180551216)，项目负责人：智晓东

Ciliary neurotrophic factor combined with forskolin and 3-isobutyl-1-methylxanthine induced muscle derived stem cells to differentiate into Schwann cells phenotype through cyclic adenosine monophosphate signaling pathway

Gong Chao1, Zhi Xiaodong1, 2, 3, Zhang Yuqiang1, 2, 3, Wang Chenliang1, Wang Kang1, Wang Wei1, 2, 3   

  1. 1Department of Orthopedics, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; 2Institute of Extra-orbital Sciences, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; 3Liaoning Provincial Key Laboratory of Medical Tissue Engineering, Jinzhou 121000, Liaoning Province, China
  • Received:2021-01-08 Revised:2021-02-05 Accepted:2021-07-19 Online:2022-07-08 Published:2021-12-28
  • Contact: Wang Wei, MD, Chief physician, Professor, Department of Orthopedics, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; Institute of Extra-orbital Sciences, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; Liaoning Provincial Key Laboratory of Medical Tissue Engineering, Jinzhou 121000, Liaoning Province, China
  • About author:Gong Chao, Master candidate, Department of Orthopedics, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China
  • Supported by:
    the School-Enterprise Cooperation Fund Project of Jinzhou Medical University, No. 2020002 (to WW); Science and Technology Foundation of Liaoning Provincial Department of Science and Technology, No. 20180551216 (to ZXD)

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摘要：

文题释义：
肌源性干细胞：是存在于骨骼肌纤维基底膜与肌膜之间的具有干/祖细胞特性的所有细胞的统称，具有来源丰富、获取容易以及在氧化和缺氧环境下仍具有出色生存能力等特性。
周围神经损伤：是一种主要由创伤、肿瘤及代谢性疾病等因素引起的临床常见病，经常会导致机体受累节段运动、感觉和自主功能的部分或全部丧失，以及出现顽固性神经痛，这严重影响患者的生活质量，并给家庭和社会造成了沉重的负担。

背景：许旺细胞是周围神经组织工程最理想的种子细胞，但许旺细胞存在诸多缺陷，因此目前的研究主要集中在具有许旺细胞特性的其他细胞上，并且许旺细胞样细胞已成为周围神经损伤修复的理想细胞来源。
目的：探讨睫状神经营养因子复合毛喉素和3-异丁基-1-甲基黄嘌呤(Fsk-IBMX)诱导肌源性干细胞分化为许旺细胞表型的方法及该诱导过程与环磷酸腺苷信号通路的相关性，以期为进一步将肌源性干细胞应用于周围神经损伤修复奠定基础。
方法：采用混合消化酶从1周龄SD乳鼠中提取肌源性干细胞，以睫状神经营养因子诱导肌源性干细胞去分化，再用Fsk-IBMX对去分化肌源性干细胞进行诱导。通过细胞形态学、细胞免疫荧光、CCK-8、Western blot、RT-qPCR等技术对肌源性干细胞、去分化细胞和许旺细胞样细胞进行鉴定及相关分析，同时检测去分化过程和许旺细胞样细胞分化过程中环磷酸腺苷信号通路相关蛋白的表达。
结果与结论：①细胞免疫荧光染色显示取自SD乳鼠的原代细胞能够被肌源性干细胞特异性标志物Desmin标记；②CCK-8结果显示第1-3代肌源性干细胞增殖活性逐渐增加，第3-8代肌源性干细胞增殖活性趋于稳定；③Western blot结果显示睫状神经营养因子诱导后成肌分化相关蛋白p21和MyoG表达降低，环磷酸腺苷信号通路相关蛋白p-CREB表达增加，Fsk-IBMX诱导后许旺细胞标志物S100β、GFAP和p75以及p-CREB表达均增加，而环磷酸腺苷抑制剂SQ22536可以抑制睫状神经营养因子和Fsk-IBMX的作用；④RT-qPCR结果与Western blot结果一致，即Fsk-IBMX诱导后S100β、GFAP和p75 mRNA表达水平增加，而SQ22536可以抑制这一效应；CCK-8结果显示Fsk-IBMX诱导后细胞活性有所降低；⑤结果显示：睫状神经营养因子复合Fsk-IBMX可以诱导肌源性干细胞分化为许旺细胞表型，并且诱导过程激活了环磷酸腺苷信号通路。
缩略语：环磷酸腺苷：cyclic adenosine monophosphate，cAMP；毛喉素：forskolin，Fsk；3-异丁基-1-甲基黄嘌呤：3-Isobutyl-1-methylxanthine，IBMX

https://orcid.org/0000-0002-9487-5427 (龚超) 

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: 干细胞, 肌源性干细胞, 睫状神经营养因子, 毛喉素, 3-异丁基-1-甲基黄嘌呤, 许旺细胞, 环磷酸腺苷, 信号通路

Abstract: BACKGROUND: Schwann cells are the most ideal seed cells for peripheral nerve tissue engineering, but Schwann cells have many defects, so the research of cell therapy to repair peripheral nerve injury mainly focuses on other cells with Schwann cells characteristics, and Schwann cells-like cells have become the main source of choice for peripheral nerve injury repair.  
OBJECTIVE: To investigate the method of differentiation of muscle derived stem cells into Schwann cells phenotypes induced by ciliary neurotrophic factor combined with forskolin and 3-isobutyl-1-methylxanthine and the correlation between the induction and the cyclic adenosine monophosphate signaling pathway, so as to lay the foundation for further application of muscle derived stem cells in peripheral nerve injury repair.
METHODS:  Muscle derived stem cells were extracted from one-week old SD rats with mixed digestive enzymes. Dedifferentiation of muscle derived stem cells was induced by ciliary neurotrophic factor, and then the dedifferentiated muscle derived stem cells were induced by 3-isobutyl-1-methylxanthine. Cell morphology, immunofluorescence, CCK-8 assay, western blot assay, RT-qPCR and other techniques were used to identify and analyze muscle derived stem cells, dedifferentiated cells and Schwann cells-like cells. The expression of proteins related to cyclic adenosine monophosphate signaling pathway during dedifferentiation and Schwann cells-like differentiation was detected.  
RESULTS AND CONCLUSION: (1) Cell immunofluorescence staining showed that primary cells taken from SD rats could be labeled with Desmin, a specific marker of muscle derived stem cells. CCK-8 assay results showed that the proliferation activity of P1-3 muscle derived stem cells gradually increased, and the proliferation activity of P3-8 muscle derived stem cells tended to stabilize. (3) Western blot assay showed that the expression of myogenic differentiation-related proteins p21 and MyoG decreased after ciliary neurotrophic factor induction, and the expression of cyclic adenosine monophosphate-related protein p-CREB increased. After 3-isobutyl-1-methylxanthine induction, the expression levels of Schwann cells markers S100β, GFAP, p75 and p-CREB increased, while cyclic adenosine monophosphate inhibitor SQ22536 could inhibit the effects of ciliary neurotrophic factor and 3-isobutyl-1-methylxanthine. (4) The RT-qPCR results were consistent with the western blot results; that was, the expression levels of S100β, GFAP and p75 mRNA increased after 3-isobutyl-1-methylxanthine induction, and SQ22536 could inhibit this effect. The results of CCK-8 assay showed that the cell activity was reduced after 3-isobutyl-1-methylxanthine induction. (5) Results concluded that ciliary neurotrophic factor combined with 3-isobutyl-1-methylxanthine can induce muscle derived stem cells to differentiate into Schwann cells phenotype, and the induction process activates the cyclic adenosine monophosphate signaling pathway.

Key words: stem cells, muscle derived stem cells, ciliary neurotrophic factor, forskolin, 3-isobutyl-1-methylxanthine, Schwann cells, cyclic adenosine monophosphate, signaling pathway

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