Department of Rehabilitation Medicine, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Department of Orthopedics, The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.
Stem Cell Res Ther. 2021 Jun 29;12(1):372. doi: 10.1186/s13287-021-02452-0.
Manipulation of neural stem and progenitor cells (NSPCs) is critical for the successful treatment of spinal cord injury (SCI) by NSPC transplantation, since their differentiation into neurons and oligodendrocytes can be inhibited by factors present in inflamed myelin. In this study, we examined the effects of LINGO-1 on spinal cord-derived NSPC (sp-NSPC) differentiation, the underlying mechanisms of action, and the functional recovery of mice after transplantation of manipulated cells.
sp-NSPCs were harvested from female adult C57/BL6 mice after SCI induced with an NYU impactor. These cells were infected with lentiviral vectors containing LINGO-1 shRNA sequence or a scrambled control and transplanted into SCI mice. Tuj-1- and GFAP-positive cells were assessed by immunofluorescence staining. Wnt5a, p-JNK, JNK, and β-catenin expression was determined by Western blot and RT-qPCR. miRNAs were sequenced to detect changes in miRNA expression. Motor function was evaluated 0-35 days post-surgery by means of the Basso Mouse Scale (BMS) and by the rotarod performance test.
We discovered that LINGO-1 shRNA increased neuronal differentiation of sp-NSPCs while decreasing astrocyte differentiation. These effects were accompanied by elevated Wnt5a protein expression, but unexpectedly, no changes in Wnt5a mRNA levels. miRNA-sequence analysis demonstrated that miR-15b-3p was a downstream mediator of LINGO-1 which suppressed Wnt5a expression. Transplantation of LINGO-1 shRNA-treated sp-NSPCs into SCI mice promoted neural differentiation, wound compaction, and motor function recovery.
LINGO-1 shRNA promotes neural differentiation of sp-NSPCs and Wnt5a expression, probably by downregulating miR-15b-3p. Transplantation of LINGO-1 shRNA-treated NSPCs promotes recovery of motor function after SCI, highlighting its potential as a target for SCI treatment.
通过神经干细胞和祖细胞（NSPCs）移植来治疗脊髓损伤（SCI），对 NSPC 的神经调控至关重要，因为它们向神经元和少突胶质细胞分化会受到炎症髓磷脂中存在的因子的抑制。在这项研究中，我们研究了 LINGO-1 对脊髓源性 NSPC（sp-NSPC）分化的影响，其作用机制以及操纵细胞移植后小鼠的功能恢复情况。
SCI 后，从雌性成年 C57/BL6 小鼠中提取 sp-NSPC。这些细胞被感染携带 LINGO-1 shRNA 序列或乱序对照的慢病毒载体，并移植到 SCI 小鼠中。通过免疫荧光染色评估 Tuj-1 和 GFAP 阳性细胞。通过 Western blot 和 RT-qPCR 测定 Wnt5a、p-JNK、JNK 和 β-catenin 的表达。通过测序检测 miRNA 表达的变化。术后 0-35 天通过 Basso 小鼠量表（BMS）和转棒性能测试评估运动功能。
我们发现 LINGO-1 shRNA 增加了 sp-NSPC 的神经元分化，同时减少了星形胶质细胞分化。这些作用伴随着 Wnt5a 蛋白表达的增加，但出人意料的是，Wnt5a mRNA 水平没有变化。miRNA 序列分析表明，miR-15b-3p 是 LINGO-1 的下游调节因子，可抑制 Wnt5a 表达。将 LINGO-1 shRNA 处理的 sp-NSPC 移植到 SCI 小鼠中，促进了神经分化、伤口压缩和运动功能恢复。
LINGO-1 shRNA 促进 sp-NSPC 的神经分化和 Wnt5a 表达，可能通过下调 miR-15b-3p 实现。移植 LINGO-1 shRNA 处理的 NSPC 可促进 SCI 后运动功能的恢复，突出了其作为 SCI 治疗靶点的潜力。
