外置磁场下静脉注射经SPIONs标记的Ngb转染ADSCs靶向治疗大鼠脊髓损伤的实验研究

Experimental Study on Intravenous Injection of ADSCs Transfected with SPIONs Labeled Ngb in External Magnetic Field for Treatment of Spinal Cord Injury in Rats

目的探讨外置磁场下静脉注射经超顺磁性氧化铁纳米颗粒标记的脑红蛋白(Ngb)转染脂肪源性干细胞(ADSCs)靶向治疗大鼠脊髓损伤的疗效。方法将36只12周龄雌、雄不均的SD大鼠用改良Allen′s法建立大鼠胸腰段(T_(11))脊髓损伤模型,依据随机数字法分为空白对照组、干细胞组、转染干细胞组,每组12只。将造模成功后的空白对照组、干细胞组及转染干细胞组实验大鼠放入0.32 T的磁场强度中,分别通过尾静脉给予DMEM高糖培养基、ADSCs及Ngb转染的ADSCs,每日1次,共7次,分别在第1、3、5、7、14、28天对各组大鼠进行BBB脊髓损伤行为学评分,在第28天处死各组大鼠取出脊髓组织进行苏木精-伊红染色、蛋白质印迹法检测及透射电镜检查。结果第5天、7天、14天、28天,干细胞组和转染干细胞组BBB功能评分均高于空白对照组(P<0.05),转染干细胞组BBB评分高于干细胞组(P<0.05);空白对照组、干细胞组、转染干细胞组Ngb表达水平分别为(0.14±0.04)mg/kg、(0.38±0.11)mg/kg、(0.72±0.05)mg/kg,其中干细胞组、转染干细胞组高于空白对照组(P<0.05),转染干细胞组高于干细胞组(P<0.05)。空白对照组透射电镜检测脊髓损伤组织突触及神经轴突生长较少,髓鞘病变严重;转染干细胞组突触及神经轴突生长最多,神经元分布广,新髓鞘发生多;干细胞组突触及神经轴突生长相对较多,髓鞘病变相对较轻;与空白对照组相比,干细胞组及转染干细胞组大鼠脊髓损伤神经功能恢复有一定改善。结论静脉注射ADSCs及Ngb基因转染的ADSCs治疗大鼠脊髓损伤均有疗效,而Ngb基因转染的ADSCs治疗大鼠脊髓损伤的疗效优于单纯使用ADSCs。

Objective To investigate the therapeutic effect of intravenous injection of superparamagnetic iron oxide nanoparticles labeled neuroglobin(Ngb)transfected adipose derived stem cells(ADSCs)in external magnetic field on spinal cord injury in rats.Methods Thirty-six SD rats of 12 weeks age were randomly used to establish a rat model of thoracolumbar(T_(11))spinal cord injury using the modified Allen′s method.They were divided into three groups according to random number method:a blank control group,a stem cell group and a transfected stem cell group,12 in each group.After successful modeling,three groups were placed in a magnetic field strength of 0.32 T,DMEM high glucose medium,ADSCs and Ngb-transfected ADSCs were given through caudal veins,once a day,for a total of 7 times.Then,ethology of injury BBB scoring was completed in the rats of each group on day 1,day 3,day 5,day 7,day 14 and day 28.Finally on day 28,all the rats were sacrificed and their spinal cord tissues were taken out for hematoxylin-eosin staining,Western blotting test and transmission electron microscopy.Results The BBB functional scores of the stem cell group and the transfected stem cell group were higher than that of the blank control group on day 5,7,14 and 28(P<0.05),and the BBB score of the transfected stem cell group was higher than that of the stem cell group(P<0.05).The expression level of Ngb in the blank control group,stem cell group and transfected stem cell group were(0.14±0.04)mg/kg,(0.38±0.11)mg/kg and(0.72±0.05)mg/kg,and the expression level of Ngb in the stem cell group and transfected stem cell group was higher than that in the blank control group(P<0.05),and that in transfected stem cell group was higher than that in stem cell group(P<0.05).In the blank control group,the growth of synapses and axons was less and the myelin sheath lesions were more serious.In the transfected stem cell group,synapses and axons grew most,neurons were distributed widely,and new myelin sheath occurred more.In the stem cell group,the growth of synapses and axons was more and myelin lesions were less.Compared with the blank control group,the nerve function recovery in the stem cell group and the transfected stem cell group was improved to some extent.Conclusion Intravenous ADSCs and Ngb transfected ADSCs were effective in the treatment of spinal cord injury in rats,and the therapeutic effect of ADSCs transfected with Ngb gene on rat spinal cord injury was better than that of ADSCs alone.