基质细胞来源因子1及其受体CXCR4参与骨髓基质细胞向脊髓全横断损伤区的迁移

Chemokine stromal cell-derived factor-1 and its receptor CXCR4 mediate migration of marrow stromal cells into the lesion site of completely transected spinal cord

背景:骨髓基质细胞具有体内、外迁移特性,但目前关于其迁移的具体机制还不十分清楚。目的:探讨基质细胞来源因子1及其受体CXCR4在骨髓基质细胞体内、外迁移中的作用。设计、时间及地点:细胞学体内实验,于2007-03/06在新加坡国立大学解剖系完成。材料:清洁级Wistar新生大鼠1只,用于骨髓基质细胞培养。清洁级成年Wistar大鼠40只,随机分为损伤模型组、假手术组,20只/组。方法:骨髓基质细胞体外趋化实验在48孔Boyden小室上进行,取25μL基质细胞来源因子1,分别以5,50,500μg/L质量浓度加到趋化板的下层,以8μm孔径的聚碳酸酯膜覆盖;并设立空白对照,单纯添加DMEM条件培养基。以含牛血清白蛋白的DMEM条件培养基调整细胞浓度至1.5×109L-1,50μL细胞悬液加到Boyden小室上层,37℃、CO2培养箱培养10h。损伤模型组大鼠制备脊髓全横断损伤,假手术组大鼠只打开椎板。脊髓全横断后1h,将5-(6-)羟基荧光素双乙酸琥珀酰亚胺酯荧光标记的骨髓基质细胞悬液1.0mL(1×109L-1)经颈内静脉注入体内。主要观察指标:免疫荧光组化染色观察骨髓基质细胞趋化因子受体CXCR4的表达,基质细胞来源因子1体外对骨髓基质细胞的趋化迁移作用,Real-timePCR定量检测脊髓损伤区基质细胞来源因子1RNA的表达,荧光显微镜下观察骨髓基质细胞向脊髓损伤区的体内迁移。结果:纯化的骨髓基质细胞呈CXCR4阳性。与空白对照比较,5,50,500μg/L基质细胞来源因子1均可明显促进骨髓基质细胞的体外趋化迁移(P<0.05),且质量浓度为50μg/L时趋化作用达峰值。与假手术组比较,损伤模型组造模后7d基质细胞来源因子1RNA的表达明显升高(P<0.05),14d时恢复至正常水平。细胞注射2周后与假手术组比较,损伤模型组损伤区迁移细胞数明显增加(P<0.05)。结论:基质细胞来源因子1体内、外可趋化骨髓基质细胞迁移,基质细胞来源因子1及其受体CXCR4参与骨髓基质细胞向脊髓全横断损伤区的迁移。

BACKGROUND： Marrow stromal cells （MSCs） own the characteristic of migration. However, the mechanisms underlying the migration of these cells remain unclear. OBJECTIVE： To explore the roles of stromal cell-derived factor-1 （SDF-1） and its receptor CXCR4 in trafficking of MSCs migration. DESIGN, TIME AND SETTING： The in vivo cytology experiment was performed at Department of Anatomy, National University of Singapore from March 2007 to June 2007. MATERIALS： MSCs were isolated and purified from a Wistar neonatal rat. Forty adult female Wistar rats were randomly divided into sham operation and experimental groups, with 20 animals in each group. METHODS： The chemotaxis assay was performed at a 48 well Boyden chamber, and a total of 25 μL SDF-1 was added to the lower layer of chamber, covered with 8 μm polycarbonate membrane filter; SDF-1 cultured in DMEM conditioned medium was served as a blank control group. Cell concentration was regulated to 1.5×10^9/L. 50 pL and cell suspension was added into the upper layer of chamber, cultured at CO2 incubator with temperature of 37 ℃ for 10 hours. Rats in the experimental group were prepared for transected spinal cord injury models, and in the sham operation group, only the vertebral plate was opened. 1.0 mL （1 × 10^9/L） MSCs suspension labeled with 5-（and-6）-carboxyfluorescein diacetate succinimidyl ester （CFDA-SE） was injected through internal jugular vein at 1 hour after completely transected spinal cord. MAIN OUTCOME MEASURES： Expression of chemokine receptor CXCR4 in MSCs, as well as the effect of SDF-1 on the migration of MSCs was observed by immunofluorescence, change of SDF-1 in lesion site of spinal cord was detected by real-time PCR analysis, as well as the in vivo migration of intravenously injected MSCs was detected by fluorescence microscopy. RESULTS： The purified MSCs were positive to CXCR4. Compared to the blank control group, SDF-1 with concentrations of 5, 50, and 500 μg/L could accelerated the migration of MSCs （P 〈 0.05）, which reached a peak with concentration of 500 pg/L. The expression of SDF-1 RNA was obvious increased in the experimental group than that of the sham operation group （P 〈 0.05）, and returned to a normal level at 14 days. At 2 weeks after cell injection, the number of MSCs migrated to the lesion site of completely transected spinal cord was significant increased than sham operation group （P 〈 0.05）. CONCLUSION： SDF-1 may contribute to MSCs migration in vitro and in vivo. SDF-1 and its receptor CXCR4 are involved in the migration of injected MSCs to the lesion site of completely transected spinal cord.