游离NgR促进新生大鼠背根神经节突起体外生长

目的 观察生物合成的游离NgR对新生大鼠原代背根神经节(DRG)神经元突起生长的影响.方法 以ViraPowerTM慢病毒将NgR(310)ecto基因转染人培养的骨髓基质细胞,收集培养上清获得游离NgR.分离新生大鼠DRG神经元并分为两组,对照组直接将神经元种植到经含中枢神经系统(CNS)髓鞘蛋白包被的培养皿中培养;实验组先将骨髓基质细胞培养上清加入含CNS髓鞘蛋白的培养皿中,孵育4 h后再植入DRG神经元.两组细胞均在培养24 h后固定,进行βⅢ-tubulin免疫组织化学染色,图像观察神经元及其突起生长情况.结果 基因修饰的骨髓基质细胞,转染后48 h间接免疫荧光的方法检测可见胞浆内荧光表达.种植24 h后对照组DRG细胞很少有突起长出;而实验组DRG细胞中60%的神经元长出突起,且突起较长.结论 游离NgR能够竞争性结合脊髓损伤后局部分泌的轴突生长抑制因子,从而保护生理性NgR,可能是促轴突再生和脊髓功能恢复的一种新策略.

Role of soluble NgR in promotion of DRG neurite sprouting in vitro in rats

Objective To observe the effect of soluble NgR on rat DRG neurite sprouting in vitro. Methods Gene encoding NgR(310)ecto was transduced into bone marrow stromal cells (BM-SCs). Soluble NgR was obtained from the supernatant of BMSCs culture. DRG neurons of neonate rat were isolated and allocated into two groups, ie, control group (DRG neurons were cultured in slides coa-ted with myelin protein of spinal cord) and experiment group (the slides were pre-incubated with the su-pernatant of BMSCs for four hours). BMSCs were immobilized after 24 hours of culture. Immunohis-tostaining with βⅢ-tubulin and fluorescence microscope were employed to analyze the neurite growth.Results Positive staining of intracytoplasm was found with indirect fluorescence microscope in the BM-SCs 48 hours following transduction. Little neurite was found sprout to DRG neurons in the control group 24 hours after transduction. In contrast, longer neurite was observed in about 60% neurons in the experi-ment group. Conclusion Soluble NgR can competitively bind to the inhibitory factors secreted local-ly, protect physiological NgR and promote axonal regeneration and functional recovery of the spinal cord following spinal cord injury.