胚胎脊髓干细胞移植联合神经生长因子对成鼠损伤脊髓结构和功能恢复的影响

Influence of transplantation of embryonic spinal cord stem cell combined with nerve growth factor on injured spinal structure and recovery of function in adult rats

目的:将胚胎脊髓干细胞移植及神经生长因子联合应用于实验性脊髓损伤大鼠,观察二者联用对损伤脊髓结构和功能恢复的干预。方法:实验于2003-05/2004-02在大庆市第四医院骨科完成。选取健康8月龄SD雄鼠2只,雌鼠4只,同笼过夜,次晨观察雌鼠,发现鼠精子者确定为孕鼠并记为妊娠0d,取胚鼠6只作为供体。另选取清洁级健康雄性SD大鼠50只,随机分为5组:神经生长因子+胚胎脊髓干细胞组、神经生长因子组、胚胎脊髓干细胞组、模型对照组、空白对照组,10只/组。其中神经生长因子+胚胎脊髓干细胞组、胚胎脊髓干细胞组大鼠作为受体。①除空白对照组外,其余各组均建立T12脊髓右半切损伤动物模型。②孕鼠于妊娠14d麻醉后暴露子宫,手术显微镜下取出胚鼠,剪去头尾,移取长约3mm的胚胎脊髓,浸泡于2000Bu/mL神经生长因子液后,立即分别移植入神经生长因子+胚胎脊髓干细胞组及胚胎脊髓干细胞组刚完成的急性脊髓右半切损伤部位。神经生长因子+胚胎脊髓干细胞组、神经生长因子组均在蛛网膜下腔插入内径0.6mm、外径1mm的导管,固定后于术后每天经导管给予神经生长因子500Bu,连续2周。模型对照组与空白对照组不给予任何治疗。③术后观察各组动物的一般状态及运动功能变化。Bieschowsky还原银染色法对神经纤维进行病理观察,并对各组损伤区脊髓横断面神经纤维数进行统计。结果:实验选取SD大鼠50只,术后1周内死亡11只,除空白对照组未死亡外,模型对照组死亡2只,其余3组各死亡3只。①术后不同时间各组大鼠的一般状态:空白对照组在手术前后均未出现尿便异常情况。神经生长因子+胚胎脊髓干细胞组、神经生长因子组、胚胎脊髓干细胞组、模型对照组大鼠术后1周内均出现尿便失禁,模型对照组死亡2只,其余3组各死亡3只;术后2周有18只大鼠排尿排便功能逐渐恢复,神经生长因子+胚胎脊髓干细胞组7只,神经生长因子组4只,胚胎脊髓干细胞组5只,模型对照组2只;术后8周有20只大鼠排尿排便功能已完全恢复,神经生长因子+胚胎脊髓干细胞组7只,神经生长因子组5只,胚胎脊髓干细胞组6只,模型对照组2只。②术后不同时间各组大鼠的运动功能改变:空白对照组在手术前后感觉运动功能无异常改变。神经生长因子+胚胎脊髓干细胞组、神经生长因子组、胚胎脊髓干细胞组、模型对照组建立T12脊髓右半切损伤模型后,大鼠右后肢运动功能全部消失,左后肢出现感觉障碍;术后8周时神经生长因子+胚胎脊髓干细胞组有6只大鼠出现刺激左后肢的逃避反应,但右后肢运动功能未见改善;胚胎脊髓干细胞组有4只大鼠对左后肢痛觉刺激出现逃避反应。神经生长因子组、模型对照组大鼠右后肢常处于拖行状态,感觉与运动功能均无改善。③术后8周各组组织病理染色观察结果:神经生长因子+胚胎脊髓干细胞组与神经生长因子组、胚胎脊髓干细胞组、模型对照组之间差异有显著性意义(27.6±4.3),(4.31±0.5),(14.2±1.6),0,P<0.05)。结论:胚胎脊髓干细胞为宿主脊髓损伤区提供了优良的微环境及支持引导作用,并通过自身的分化再生与宿主神经元形成广泛联系,同时产生更多的神经营养因子,与胚胎脊髓干细胞移植联合应用可协同促进宿主脊髓功能的恢复。

AIM： The association between transplantation of fetal spinal cord stem cell and nerve growth factor （NGF） is used in the treatment of experimental spinal cord injury rats, to observe the intervention of the association in injured spinal cord structure and functional recovery. METHODS： The experiment was conducted at the Department of Orthopaedics, Daqing Municipal Fourth Hospital between May 2003 and February 2004. Two male healthy SD rats aged 8 months and 4 females were taken in the cage overnight, examining the females the second day, marking those who pregnanced as 0 day, taking 6 fetal rats as the donor. Fifty clearing healthy male SD rats were selected and divided randomly into 5 groups： NGF＋ embryonic spinal cord stem cell group （NGF＋ESS group）, NGF group, ESS group, model control group and blank control group with 10 rats in each group, and the rats in the NGF＋ESS group and ESS group were donors. ①Except blank control group, animal models were established at the right half of T12 spinal cord of each rat. ②Wombs were exposed of pregnant rats at the 14^th day after cyesis and anesthesia. Fetal rats were got under microscope; head and tail were cut. 3 mm fetal spinal was taken out for transplanting into the acute right half injured part of rats in the NGF＋ESS group and ESS group after soaping into 2 000 Bu/mL NGF liquor. A duct with the inner diameter of 0.6 mm and outside diameter of 1 mm was inserted into inferior cava of arachnoid in the NGF＋ ESS group and NGF group. The NGF 500 Bu was given each day after fixation for 2 weeks. The rats in the model control group and the blank control group were not given any therapy. ③General status and changes of motor function of animals in every group were observed after operation. Pathological observation was performed on neural fibers with Bieschowsky reduction silver staining and the number of neural fibers at cross section of spinal cord at injured region in every group was calculated. RESULTS： Eleven of 50 SD experimental rats were dead in one week after the operation. There were no death except the blank control group, and 2 rats died in the model control group and 3 rats died in other three groups. ①The general states of rats in every group at different time after operation： No abnormal emiction happened in the blank control group before and after operation. Incontinence of emiction appeared within 1 week after operation in rats of the NGF＋ESS group, NGF group, ESS group and model control group. Two rats died in the model control group, and 3 rats died in the other three groups. Function of miction and defecation recovered gradually in 18 rats at 2nd week after operation, that was 7 rats in the NGF＋ESS group, 4 rats in the NGF group, 5 rats in the ESS group and 2 rats in the model control group. At the 8th week after operation, function of miction and defecation recovered completely in 20 rats, that was 7 rats in the NGF＋ESS group, 5 rats in the NGF group, 6 rats in the ESS group and 2 rats in the model control group. ②Change of motor function of rats in every group at different time after operation： No abnormal change of function of sensofimotor in the blank control group before and after operation. After establishing fight half injured models at T12 Spinal cord, motor function of fight hindlimb of rats disappeared completely and sensory disturbance appeared at left hindlimb of rats in the NGF＋ESS group, NGF group, ESS group and model control group. At the 8^th week after operation, escaped reaction at left hindlimb appeared in 6 rats in the NGF ＋ESS group, but motor function was not improved at the fight hindlimb. Escaped reaction at left hindlimb appeared in .4 rats in the ESS group. Trailing status appeared at the fight hindlimb of rats in the NGF group and model control group, and the sensation and motor function were not improved. ③Observational result of histopathologieal staining in every group at the 8^th week after operation： It in the NGF＋ESS group had significant difference with that in the NGF group, ESS group and model control group （27.6±4.3）, （4.31±0.5）, （14.2±1.6） ,0,P 〈 0.05）. CONCLUSION： The ESS provides fine microenvironment and supporting bootstrapping effect for host spinal injury region and form extensive contact with host neuron via differentiation and regeneration of themselves. Meanwhile, multiple neurotrophic factors appear, and the associated application with ESS transplantation can accelerate synergistically the recovery of host spinal function.