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目的 BMSCs作为雪旺细胞的替代细胞可提高周围神经损伤修复效果,但目前对于神经支架内添加适宜的种子细胞数量未达成共识。研究不同数量BMSCs对大鼠背根神经节(dorsal root ganglion,DRG)生长的影响。方法取4周龄雄性SD大鼠3只,体重80～100 g,体外培养扩增BMSCs。取新生1～2日龄SD大鼠3只,雌雄不限,体重4～6 g,制备DRG。取第3代BMSCs制备BMSCs-生物蛋白胶复合物,按照加入细胞数量的不同,将实验分为A组(1×103个)、B组(1×104个)、C组(1×105个)和D组(0个),并与SD大鼠DRG共培养。48 h后通过形态学观察,神经丝蛋白200和雪旺细胞S-100免疫荧光染色,对SD大鼠DRG轴突生长长度、雪旺细胞迁移距离和轴突面积指数进行定量评价。结果 48 h后可见各组BMSCs生长出多个长突起;雪旺细胞移行和轴突从DRG长出,呈多方向性生长;BMSCs生物蛋白胶具有生物活性且影响DRG生长。A、B、C组DRG轴突生长长度及雪旺细胞迁移距离均明显大于D组(P<0.05),C组小于B组(P<0.05),A、C组间及A、B组间差异无统计学意义(P>0.05)。A、B组轴突面积指数明显大于D组(P<0.05),C组大于D组但差异无统计学意义(P>0.05),A、B、C组间差异无统计学意义(P>0.05)。结论乳鼠DRG体外培养实验,可作为研究周围神经损伤修复的体外模型。不同数量BMSCs生物蛋白胶复合物对DRG生长的影响具有量效关系,为组织工程神经选用合适数量BMSCs提供了理论依据。
The purpose of BMSCs as a substitute for Schwann cells can improve the repair effect of peripheral nerve injury, but currently there is no consensus on the appropriate number of seed cells in neural scaffolds. To study the effects of different numbers of BMSCs on the growth of rat dorsal root ganglion (DRG). Methods Three 3-week-old male Sprague-Dawley rats weighing 80-100 g were used in this study. BMSCs were cultured and expanded in vitro. Take freshmen 1 to 2 days old SD rats 3, male or female, weighing 4 ~ 6 g, preparation of DRG. The 3rd generation BMSCs were used to prepare BMSCs-bioprotein composite. According to the number of cells added, the experiment was divided into group A (1 × 103), group B (1 × 104), group C (1 × 105 ) And D group (0), and co-cultured with SD rat DRG. After 48 h, the length of DRG axon, the migration distance of Schwann cells and the axonal area index were quantitatively evaluated by morphological observation, neurofilament protein 200 and Schwann cell S-100 immunofluorescence staining. Results After 48 h, many long neurites were found in BMSCs. Schwann cells migrated and axons grew out of DRG, which showed multi-directional growth. BMSCs bioprotein gel had biological activity and affected the growth of DRG. The length of DRG axon and the migration distance of Schwann cells in group A, B and C were significantly higher than those in group D (P <0.05), and those in group C were less than those in group B (P <0.05) The difference was not statistically significant (P> 0.05). The axial area index of group A and group B was significantly larger than that of group D (P <0.05), but the difference between group C and group D was not statistically significant (P> 0.05). There was no significant difference between groups A, B and C (P> 0.05). Conclusion The in vitro culture of suckling rat DRG can be used as an in vitro model to study the repair of peripheral nerve injury. The effect of different amounts of BMSCs on the growth of DRG has a dose-effect relationship and provides a theoretical basis for the selection of the appropriate number of BMSCs for tissue engineering nerves.