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  

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

作　　者：龚超 智晓东 张玉强 王晨亮 王康 王伟 Gong Chao;Zhi Xiaodong;Zhang Yuqiang;Wang Chenliang;Wang Kang;Wang Wei(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)

机构地区：[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

出　　处：《中国组织工程研究》2022年第19期2970-2977,共8页Chinese Journal of Tissue Engineering Research

基　　金：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。

摘　　要：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;(2)CCK-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;(3)Western 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;(4)RT-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。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 differentiationrelated proteins p21 and MyoG decreased after ciliary neurotrophic factor induction,and the expression of cyclic adenosine monophosphate-relat

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 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]