MCP-1／CCR2通路参与骨髓基质细胞向脊髓全横断损伤区迁移的实验研究

MCP-1/CCR2 Pathway Mediates Migration of Marrow Stromal Cells into Lesion Site of Completely Transected Spinal Cord

【目的】探讨单核细胞趋化蛋白-1(MCP-1)及其受体CCR2在骨髓基质细胞(MSC)体内、外迁移中的作用。【方法】体外培养、纯化大鼠MSC,取第五代MSC行免疫荧光鉴定;免疫组化、RT-PCR检测纯化MSC表达CCR2情况;Boyden小室法检测趋化因子MCP-1(5～500ng/mL)对MSC的趋化迁移作用及其特异性。42只成年大鼠用于体内研究(脊髓全横断组24只,假手术组9只,正常大鼠9只),分别于术后1,3,7,14d取材,行免疫组化检测MCP-1表达,或行MCP-1的Real-timePCR定量分析,或颈内静脉注射荧光标记的MSC,观察MSC向脊髓迁移情况。【结果】第5代MSC都表达间充质干细胞标记物Vimentin、Laminin及Fibronectin;细胞免疫荧光、RT-PCR证实MSC表达趋化因子受体CCR2;MCP-1(5～500ng/mL)体外可趋化MSC迁移(P﹤0.05),抗MCP-1抗体可对抗其趋化迁移作用(P﹤0.05)。脊髓全横断组、对照组脊髓均表达MCP-1,但细胞分布、染色存在差异,影响MCP-1定量。脊髓损伤后趋化因子MCP-1RNA在1d、3d及7d较对正常对照组差异有统计学意义(P<0.05),术后14d时MCP-1RNA与正常对照相比差异无统计学意义(P>0.05),伴随其变化,脊髓损伤区迁移MSC较对照差异有统计学意义(P﹤0.05)。【结论】MCP-1体内、外可趋化MSC迁移,MCP-1/CCR2通路参与MSC向脊髓全横断损伤区的迁移。

[Objective] To explore the roles of monocyte chemoattractant protein-1 （MCP-1） and its receptor CCR2 in trafficking of marrow stromal cells （MSC） migration. [Methods] Isolated and cultured MSC for 5 passages, then using Vimentin, Laminin, and Fibronectin to identify MSC. Using immunofluorescence and RT-PCR to detect whether MSC, in vitro, express CCR2. Using chemotaxis assay （Boyden method） to detect whether MCP-1 （5 -500 ng/mL） effect MSC migration in vitro. A total of 42 adult rats were used in vivo study （24 rats for completely transected spinal cord models, 9 rats for sham operation and 9 rats for normal control）. At 1, 3, 7, and 14 days after operation ,expression of MCP-1 was detected by immunofluorescence, or real-time PCR was executed, and some rats were intravenously injected CFDA-SE labeled MSC to detect the migration of MSC in vivo. [Results] After 5 passages, all MSC express Vimentin, Laminin, and Fibronectin. Immunofluorescence and RT-PCR show MSC expression, the respective receptors for MCP-1 and CCR2. MCP-1 in vitro induce the migration of MSC （P 〈 0.05）, and which can be blocked by anti-MCP-1. MCP-1 was expressed in the spinal cord of experimental groups and control groups. The distribution and staining of MCP-1 were differential in both groups, which affected the results of quamitation of MCP-1. Real-time PCR results revealed that there was significant increase of MCP-1 at 1, 3, and 7 days after operation compared to that of the normal control （P 〈 0.05）. Subsequently, at 14 days postoperation, the expression of MCP-1 decreased to normal （P 〉 0.05）. Intravenously injected MSC migrate to the lesion site of completely transected spinal cord where MCP-1 was assayed to be increased after spinal cord injury （P 〈 0.05）. [Conclusion] MCP-I/CCR2 pathway mediates the migration of MSC into the lesion site of completely transected spinal cord.