人神经干细胞移植对缺氧缺血性脑病大鼠神经修复的研究

Research on the Neurorestorative Effects of Human Neural Stem Cell Transplantation in Rats with Hypoxic-Ischemic Encephalopathy

目的探究移植人神经干细胞(hNSCs)治疗新生大鼠中、重度缺氧缺血性脑病(HIE)及后遗症期对神经的保护机制。方法取80只7日龄雄性SD大鼠,随机取其中55只采用Rice-Vannucci法制备HIE模型,建模后24 h对存活模型鼠采用Longa评分筛选出中、重度神经损伤大鼠并随机分为HIE+PBS组(PBS组,n=23)、HIE+hNSCs组(NSC组,n=23),从剩余25只大鼠中随机取23只作为假手术组(Sham组,n=23),Sham组只游离右侧颈总动脉,不予离断,也不予缺氧处理。三组同步给予药物抗排斥反应,PBS组与NSC组在建模后3 d分别经右侧脑室注射5μL PBS溶液或hNSCs悬液。移植后10 d,三组各随机取15只大鼠采用酶联免疫吸附实验(ELISA)检测脑内血管内皮生长因子(VEGF)、脑源性神经营养因子(BDNF)含量,采用免疫荧光染色观察两种因子在脑内的分布情况。移植后12周,通过免疫荧光染色观察NSC组大鼠脑内hNSCs的迁移、分化,以水迷宫实验对三组各剩余的8只大鼠进行神经功能检测。结果移植后10 d,NSC组VEGF、BDNF两种因子含量均最多,PBS组次之,Sham组最少,NSC组两种因子含量均显著高于Sham组(均P<0.05)。移植后12周,hNSCs向HIE大鼠两侧脑半球迁移,以右侧为主,成熟神经元分化率约30%。移植后12周,水迷宫实验中,PBS组潜伏期均长于Sham组及NSC组(均P<0.05),PBS组穿越平台次数均少于Sham及NSC组(均P<0.05);而Sham组与NSC组结果比较,差异无统计学意义(均P>0.05)。结论脑室移植hNSCs通过加强HIE大鼠脑内VEGF、BDNF的旁分泌效应,及迁移并分化为神经元的替代作用,从而促进HIE大鼠后遗症期神经损伤的修复,为HIE治疗提供新思路。

Objective To explore transplanted human neural stem cells(hNSCs)for the treatment of moderate and severe hypoxic-ischemic encephalopathy(HIE)in neonatal rats and its mechanisms of neuroprotection during the sequela peri-od.Methods Eighty 7-day-old male SD rats were randomly selected.Among them,55 rats were randomly selected to make HIE model by Rice-Vannucci method.After modeling for 24 h,the surviving rats with moderate and severe injury were screened out by Longa score and randomly divided into HIE+PBS group(PBS group,n=23)and HIE+hNSCs group(NSC group,n=23).Twenty-three rats were randomly selected from the remaining 25 rats as Sham operation group(Sham group,n=23).In Sham group,there was only the isolation of the right common carotid artery without dissection or hypoxia.Simultaneous drug adminis-tration was conducted to suppress rejection reactions took place in all three groups.Three days after modeling,the PBS and NSC groups were administered with 5μL of PBS solution or hNSCs suspension via right intraventricular transplantation,respective-ly.Ten days post-transplantation,15 rats from each group were randomly selected for an enzyme-linked immunosorbent assay(ELISA)to measure the levels of vascular endothelial growth factor(VEGF)and brain-derived neurotrophic factor(BDNF),im-munofluorescent staining was used to observe the distribution of the two factors.Twelve weeks after transplantation,immuno-fluorescent staining was used to observe the migration and differentiation of hNSCs in the brains of the NSC group rats,and the remaining 8 rats from each group underwent a water maze test to assess neural function.Results Ten days after transplanta-tion,the levels of VEGF and BDNF were the highest in the NSC group,followed by the PBS group and the Sham group,and the levels of both factors were higher in the NSC group than in the Sham group(all P<o.05).Twelve weeks after transplantation,hNSCs migrated to both hemispheres of HIE rats,mainly to the right side,and the mature neuron differentiation rate was about 30%.Twelve weeks after transplantation,the latency of the PBS group was longer than that of the Sham and NSC groups in the water maze test,and the times of platform crossing of the PBS group were shorter than that of the Sham and NSC groups(all P<0.05),but there was no significant difference between the Sham and NSC groups(P>0.05).Conclusion Ventricular trans-plantation of hNSCs can enhance the paracrine effect of VEGF and BDNF in the brain of HIE rats and the substitution effect of migration and differentiation into neurons,thus promoting the repairment of nerve injury in the sequela of HIE rats and provi-ding new ideas for the treatment of HIE.