神经干细胞NgR基因沉默移植治疗大鼠脊髓损伤

目的 探讨是否可以通过沉默Nogo-66的受体(NgR)基因的方法提高神经干细胞(NSCs)移植对脊髓半横断损伤大鼠的治疗效果.方法 取胎龄为14～16 d的Wistar胎鼠脑组织,体外培养NSCs,经小分子干扰RNA(siRNA)转染以沉默NgR基因,用蛋白质免疫印迹法(Western blotting)检测NSCs在转染前后NgR的蛋白表达.另将36只Wistar大鼠按随机数字表法均分成3组,切断胸8、胸9段右半侧脊髓致伤.A组为注射等量不含NSCs的培养液对照组;B组为单纯NSCs移植组;C组为沉默NgR基因NSCs移植组.各组分别于伤后1、2、4、6、8周采用斜板实验进行行为学评估,观察神经恢复情况.第4周取脊髓组织制备病理切片,行苏木素-伊红(HE)染色及5-溴脱氧尿嘧啶(BrdU)免疫组化染色;8周后取脊髓组织,行辣根过氧化物酶(HRP)示踪观察.结果 转染siRNA后,NgR的蛋白表达水平较转染前明显降低(1.03±0.08比1.88±0.15,P=0.002).斜板实验:8周时,B、C两组大鼠后肢运动功能均明显恢复,C组较B组为快(P＜0.05).A组切片未见神经轴索通过,B、C组可见少量神经轴索样结构.BrdU阳性细胞及HRP阳性神经纤维束数C组＞B组＞A组(BrdU阳性细胞:A组(39.82±14.07)个、B组(91.68±12.34)个、C组(103.67±11.52)个,HRP阳性神经纤维束:A组(11.35±1.71)个、B组(39.87±2.42)个、C组(83.64±2.13)个],且各组间差异有统计学意义(均P＜0.01).结论 NSCs的NgR基因沉默后移植治疗大鼠脊髓损伤可明显改善损伤后大鼠的后肢功能.

Treatment of spinal cord injury by transplanting neural stem cells with NgR gene silencing

Objective To determine whether Nogo-66 receptor (NgR) gene silencing in neural stem cells (NSCs) can ameliorate spinal cord injury (SCI) in rats.Methods The brain tissue of Wistar rats of embryo (age 14-16 days) was obtained,and NSCs were cultured in suspension culture,and they were transfeeted by siRNA to silence the expression of NgR.Western blotting was used to assess the silencing efficiency.Thirty-six Wistar rats were divided randomly into three groups.Hemi-truncation of right-half side of 8,9 thoracic spinal cord was performed.Group A was injected same amount of culture medium without NSCs,B group was given naive NSCs suspension,and C group same amount of NgR gene silenced NSCs.At 1,2,4,6,8 weeks post-injury,the motor power of the hind limbs of all animals were evaluated with inclined plane test.The tissue of the injured portion of spinal cord was obtained for pathological examination with hematoxylin and eosin (HE) and 5-bromodeoxyuridine (BrdU) immuni-histochemistry staining after 4 weeks,and observed with horseradish peroxidase (HRP) nerve trace techniques after 8 weeks.Results Western blotting confirmed that the expression of NgR was down-regulated by transfection of siRNA at 24 hours after the transfection (1.03±0.08 vs.1.88±0.15,P=0.002).Inclined plane test showed the performance was improved in B and C groups,and that of C group surpassed that of B group after 8 weeks (P＜0.05).In A group,there was no passage of axons through the injury,while in B and C groups,there were several nerve axon-like structure in the injured part.BrdU positive cells and HRP-labeled neurofibriles in C group＞B group＞A group (BrdU positive cells; A group 39.82±14.07,B group 91.68±12.34,C group 103.67±11.52,HRP-labeled neurofibriles; A group 11.35±1.71,B group 39.87±2.42,C group 83.64±2.13),and there was statistical significance among three groups (all P＜0.01).ConclusionTransplantation of NSCs of NgR gene silencing transplants into the injured spinal cord tissue can significantly improve the neurological function in the rats.