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目的探讨间充质祖细胞(mesenchymal progenitor cells,MPCs)源性的神经元样细胞肌内移植后能否维持其自身特性。方法取3周龄健康GFP转基因C57小鼠后肢长骨进行MPC培养及鉴定,选取生长良好的第3代MPC,采用神经元原代培养上清液进行神经元样细胞诱导分化后收集细胞,并制备成5×105/μL细胞悬液备用。选取12周龄健康C57小鼠24只,按随机数字表法分为损伤+移植细胞组、损伤+生理盐水组、假手术+移植细胞组、假手术+生理盐水组。损伤+移植细胞组及假手术+移植细胞组:将制备的5μL MPC悬液散点注入小鼠三头肌内;损伤+生理盐水组和假手术+生理盐水组:同法注入等量生理盐水。术后4周取材,荧光显微镜下观察肌内移植细胞存活情况,电镜观察肌肉超微结构的变化,免疫荧光染色检测神经元特异性标志物NSE、NeuN及神经胶质特异性标志物GFAP的表达情况。结果肌内移植细胞生长良好且均匀分布于肌细胞间隙,并抑制了肌细胞核、线粒体、内质网的退变及肌肉纤维化的发生。损伤+移植细胞组定植存活的移植细胞阳性表达:NSE(58.35±1.37)%、NeuN(60.22±0.16)%及GFAP(13.32±1.65)%,损伤+生理盐水组、假手术+移植细胞组、假手术+生理盐水组均无阳性细胞表达。结论 MPC具有良好的生物学活性,经体外诱导分化为神经元及神经胶质样细胞后移植于失神经支配肌内可定植存活,并能够维持神经元及神经胶质样细胞的特性,同时有效抑制了肌细胞核、线粒体、内质网的退变及肌肉纤维化的发生。
Objective To investigate the ability of mesenchymal progenitor cells (MPCs) derived neuron-like cells to maintain their own characteristics after intramuscular transplantation. Methods The long bones of the hindlimbs of 3-week-old healthy GFP transgenic C57 mice were cultured and identified by MPC. Third-generation MPCs with good growth were selected and neuronal-derived primary culture supernatant was used for neuronal-like cell differentiation. Cells were collected and cultured Into 5 × 105 / μL cell suspension spare. Twenty-four healthy C57 mice aged 12 weeks were selected and divided into four groups according to random number table: injury + transplantation group, injury + saline group, sham operation + transplantation group and sham operation + saline group. Injury + transplantation group and sham + transplantation group: The prepared 5μL MPC suspension was injected into mice triceps innoculation; injury + saline group and sham + saline group: the same method of injecting an equal volume of saline . Survival of intramuscular transplantation cells was observed under fluorescence microscope and the ultrastructure of muscle was observed under electron microscope. The expression of NSE, NeuN and glial-specific marker GFAP were detected by immunofluorescence staining Happening. Results The intramuscular transplanted cells grew well and distributed evenly in the myocyte gap, and inhibited the degeneration of muscle cell nucleus, mitochondria and endoplasmic reticulum and the occurrence of muscle fibrosis. The positive expression of surviving transplanted cells in injured + transplanted cells group were NSE (58.35 ± 1.37)%, NeuN (60.22 ± 0.16)%, GFAP (13.32 ± 1.65)%, injury + saline group, sham operation + transplantation group, No sham operation + saline group positive cells. Conclusion MPC has good biological activity. After being differentiated into neurons and glial-like cells in vitro, MPC transplanted into the denervated innervation can be grafted to survive, and can maintain the characteristics of neurons and glial-like cells, while effective Inhibition of muscle cell nucleus, mitochondria, endoplasmic reticulum degeneration and the occurrence of muscle fibrosis.