中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (30): 4787-4792.doi: 10.12307/2022.757

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

小分子化合物毛喉素和3-异丁基-1-甲基黄嘌呤诱导骨髓间充质干细胞 分化为许旺细胞样表型的可能性

吴小松1，龚  超1，娄  盼1，王  伟2，3，4   

  1. 1荆门市第一人民医院骨科，湖北省荆门市   448000；2锦州医科大学附属第一医院骨科，辽宁省锦州市   121000；3锦州医科大学骨外科学研究所，辽宁省锦州市   121000；4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:2021-09-16 接受日期:2021-10-22 出版日期:2022-10-28 发布日期:2022-03-29
  • 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:吴小松，男，1970年生，湖北省荆门市人，汉族，1992年三峡大学毕业，副主任医师，主要从事组织损伤修复与再生研究。 龚超，男，1993年生，湖北省十堰市人，汉族，2021年锦州医科大学毕业，硕士，医师，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；荆门市一般科技计划项目(2021YFYB072)，项目负责人：娄盼

Possibility of forskolin and 3-isobutyl-1-methylxanthine in inducing bone marrow mesenchymal stem cells to differentiate into Schwann cells-like phenotype

Wu Xiaosong1, Gong Chao1, Lou Pan1, Wang Wei2, 3, 4    

  1. 1Department of Orthopedics, Jingmen No.1 People’s Hospital, Jingmen 448000, Hubei Province, China; 2Department of Orthopedics, First Affiliated Hospital, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; 3Institute of Extra-orbital Sciences, Jinzhou Medical University, Jinzhou 121000, Liaoning Province, China; 4Liaoning Provincial Key Laboratory of Medical Tissue Engineering, Jinzhou 121000, Liaoning Province, China
  • Received:2021-09-16 Accepted:2021-10-22 Online:2022-10-28 Published:2022-03-29
  • 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:Wu Xiaosong, Associate chief physician, Department of Orthopedics, Jingmen No.1 People’s Hospital, Jingmen 448000, Hubei Province, China Gong Chao, Master, Physician, Department of Orthopedics, Jingmen No.1 People’s Hospital, Jingmen 448000, Hubei Province, China
  • Supported by:
    School-Enterprise Cooperation Fund Project of Jinzhou Medical University, No. 2020002 (to WW); General Science and Technology Project of Jingmen, No. 2021YFYB072 (to LP)

PDF

43

摘要：

文题释义：
周围神经损伤：是一种主要由创伤、肿瘤及代谢性疾病(如糖尿病)等因素引起的骨科常见疾病，很难用标准的手术治疗来修复，经常面临神经再生不良、功能恢复不全等问题。
许旺细胞：是周围神经系统的主要结构和功能细胞，在损伤神经组织的华勒变性过程和再生过程中均发挥着至关重要的作用，是周围神经损伤修复与重建中最重要、也是首选的种子细胞。

背景：周围神经损伤是一种主要由创伤等因素引起的临床常见疾病，研究表明，由骨髓间充质干细胞转分化的许旺细胞可以促进周围神经损伤的修复与重建，但其诱导方法尚未达成共识，并且细胞神经转分化的机制仍尚不明确。
目的：探讨小分子化合物(毛喉素和3-异丁基-1-甲基黄嘌呤)诱导骨髓间充质干细胞分化为许旺细胞的可行性，以及该诱导过程与cAMP/PKA通路的相关性。
方法：采用全骨髓贴壁法从1周龄SD乳鼠四肢骨骨髓中提取出原代骨髓间充质干细胞，运用细胞免疫荧光对其进行鉴定，并用CCK-8法检测各代骨髓间充质干细胞在不同培养时间点的增殖活性变化，以小分子化合物对骨髓间充质干细胞进行体外诱导分化，实验分为3组：对照组用神经生长培养基处理120 h、诱导组用神经生长培养基+毛喉素+3-异丁基-1-甲基黄嘌呤处理120 h、抑制组用神经生长培养基+毛喉素+3-异丁基-1-甲基黄嘌呤+cAMP抑制剂SQ22536处理120 h，每60 h更换1次培养基。通过细胞形态学、Western blot等技术对许旺细胞样细胞进行鉴定及相关分析，同时检测分化过程中cAMP相关蛋白的表达。
结果与结论：①细胞免疫荧光染色显示分离培养的原代细胞能够被CD44和CD90标记，CCK-8结果显示在相同培养时间点，第1-3代骨髓间充质干细胞增殖活性逐渐增加，第3-6代骨髓间充质干细胞增殖活性逐渐降低；②Western blot结果显示小分子化合物诱导后许旺细胞特异性标志物S100β、GFAP和p75以及cAMP相关蛋白p-CREB表达均明显增加(P < 0.001；P < 0.001；P < 0.05；P < 0.000 1)，而cAMP/PKA抑制剂SQ22536可以抑制这一效应(P < 0.01；P < 0.01；P < 0.05；P < 0.001)；③结果表明，该小分子化合物可以诱导骨髓间充质干细胞分化为许旺细胞表型，并且诱导过程中可能激活了cAMP/PKA通路。
缩略语：毛喉素：Forskolin，Fsk；3-异丁基-1-甲基黄嘌呤：3-Isobutyl-1-methylxanthine，IBMX

https://orcid.org/0000-0002-7535-9160 (吴小松) 

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: 骨髓间充质干细胞, 毛喉素, 3-异丁基-1-甲基黄嘌呤, 许旺细胞, cAMP, PKA, 通路

Abstract: BACKGROUND: Peripheral nerve injury is a common clinical disease mainly caused by trauma and other factors. Studies have shown that Schwann cells transdifferentiated from bone marrow mesenchymal stem cells can promote the repair and reconstruction of peripheral nerve injury, but the induction method has not yet reached a consensus, and the mechanism of cell neurotransdifferentiation is still unclear.  
OBJECTIVE: To investigate the feasibility of the small molecule compounds (Forskolin and 3-isobutyl-1-methylxanthine, Fsk-IBMX, FI) induced bone marrow mesenchymal stem cells to differentiate into Schwann cells, and the correlation between the induction process and the cAMP/PKA pathway.
METHODS:  The primary bone marrow mesenchymal stem cells were extracted from the bone marrow of the limbs of 1-week-old SD rats by the whole bone marrow adherence method. The bone marrow mesenchymal stem cells were identified by cellular immunofluorescence. The changes of proliferation activity of each generation of bone marrow mesenchymal stem cells were detected by CCK-8 assay at different time points of culture. Bone marrow mesenchymal stem cells were induced by FI in vitro. The experiment contained three groups. The samples in the control group were treated with nerve growth medium for 120 hours. Those in the induction group were treated with nerve growth medium + FSK + IBMX + SQ22536 for 120 hours. Those in the inhibition group were treated with nerve growth medium + FSK + IBMX + SQ22536 treated for 120 hours. The medium should be replaced every 60 hours. Schwann cell-like cells were identified and analyzed by cell morphology and western bot assay. The expression of cAMP-related proteins during the differentiation was detected.  
RESULTS AND CONCLUSION: (1) Cell immunofluorescence staining showed that primary cells from the limb bone marrow of one-week-old SD rats could be labeled with BMSCs-specific markers CD44 and CD90. CCK-8 assay results showed that the proliferation activity of bone marrow mesenchymal stem cells increased gradually from passage 1 to passage 3 and decreased gradually from passage 3 to passage 6 at the same time point of culture. (2) Western blot assay results demonstrated that the expression of Schwann cells-specific markers S100β, GFAP, p75, and cAMP-related protein p-CREB increased significantly after FI induction (P < 0.001; P < 0.001; P < 0.05; P < 0.000 1), while the cAMP/PKA inhibitors SQ22536 could inhibit this effect (P < 0.01; P < 0.01; P < 0.05; P < 0.001). (3) It is concluded that FI can induce bone marrow mesenchymal stem cells to differentiate into Schwann cells phenotype and may activate the cAMP/PKA pathway during this induction process.

Key words: bone marrow mesenchymal stem cells, Forskolin, 3-isobutyl-1-methylxanthine, Schwann cells, cAMP, PKA, pathway

中图分类号: