miR-145a-5p 修饰的 M2 型小胶质细胞移植促进小鼠脊髓损伤组织修复。

BACKGROUND: The traumatic spinal cord injury (SCI) can cause immediate multi-faceted function loss or paralysis. Microglia, as one of tissue resident macrophages, has been reported to play a critical role in regulating inflammation response during SCI processes. And transplantation with M2 microglia into SCI mice promotes recovery of motor function. However, the M2 microglia can be easily re-educated and changed their phenotype due to the stimuli of tissue microenvironment. This study aimed to find a way to maintain the function of M2 microglia, which could exert an anti-inflammatory and pro-repair role, and further promote the repair of spinal cord injury. METHODS: To establish a standard murine spinal cord clip compression model using Dumont tying forceps. Using FACS, to sort microglia from C57BL/6 mice or CX3CR1 mice, and further culture them in vitro with different macrophage polarized medium. Also, to isolate primary microglia using density gradient centrifugation with the neonatal mice. To transfect miR-145a-5p into M2 microglia by Lipofectamine2000, and inject miR-145a-5p modified M2 microglia into the lesion sites of spinal cord for cell transplanted therapy. To evaluate the recovery of motor function in SCI mice through behavior analysis, immunofluorescence or histochemistry staining, Western blot and qRT-PCR detection. Application of reporter assay and molecular biology experiments to reveal the mechanism of miR-145a-5p modified M2 microglia therapy on SCI mice. RESULTS: With in vitro experiments, we found that miR-145a-5p was highly expressed in M2 microglia, and miR-145a-5p overexpression could suppress M1 while promote M2 microglia polarization. And then delivery of miR-145a-5p overexpressed M2 microglia into the injured spinal cord area significantly accelerated locomotive recovery as well as prevented glia scar formation and neuron damage in mice, which was even better than M2 microglia transplantation. Further mechanisms showed that overexpressed miR-145a-5p in microglia inhibited the inflammatory response and maintained M2 macrophage phenotype by targeting TLR4/NF-κB signaling. CONCLUSIONS: These findings indicate that transplantation of miR-145a-5p modified M2 microglia has more therapeutic potential for SCI than M2 microglia transplantation from epigenetic perspective.

背景：创伤性脊髓损伤（SCI）可导致即时的多方面功能丧失或瘫痪。小胶质细胞作为组织驻留巨噬细胞之一，据报道在 SCI 过程中调节炎症反应中发挥关键作用。并且将 M2 小胶质细胞移植到 SCI 小鼠中可促进运动功能的恢复。然而，由于组织微环境的刺激，M2 小胶质细胞很容易重新教育并改变其表型。本研究旨在找到一种维持 M2 小胶质细胞功能的方法，使其发挥抗炎和促进修复的作用，进一步促进脊髓损伤的修复。

方法：使用 Dumont 结扎钳建立标准的小鼠脊髓夹压迫模型。使用 FACS 从小鼠或 CX3CR1 小鼠中分离小胶质细胞，并进一步在不同的巨噬细胞极化培养基中进行体外培养。此外，使用新生小鼠的密度梯度离心法分离原代小胶质细胞。通过 Lipofectamine2000 将 miR-145a-5p 转染到 M2 小胶质细胞中，并将修饰后的 miR-145a-5p 修饰的 M2 小胶质细胞注射到脊髓损伤部位进行细胞移植治疗。通过行为分析、免疫荧光或组织化学染色、Western blot 和 qRT-PCR 检测评估 SCI 小鼠运动功能的恢复。应用报告基因检测和分子生物学实验揭示 miR-145a-5p 修饰的 M2 小胶质细胞治疗 SCI 小鼠的机制。

结果：通过体外实验，我们发现 miR-145a-5p 在 M2 小胶质细胞中高度表达，miR-145a-5p 的过表达可以抑制 M1 同时促进 M2 小胶质细胞极化。然后将过表达 miR-145a-5p 的 M2 小胶质细胞递送到损伤的脊髓区域，可显著加速运动功能的恢复，并防止小鼠的神经胶质瘢痕形成和神经元损伤，效果甚至优于 M2 小胶质细胞移植。进一步的机制表明，过表达 miR-145a-5p 在小胶质细胞中通过靶向 TLR4/NF-κB 信号通路抑制炎症反应并维持 M2 巨噬细胞表型。

结论：这些发现表明，从表观遗传学角度来看，与 M2 小胶质细胞移植相比，移植过表达 miR-145a-5p 的 M2 小胶质细胞对 SCI 具有更大的治疗潜力。