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目的观察神经营养因子与神经干细胞(NSCs)联合移植对缺氧缺血性脑损伤(HIBD)大鼠的治疗效果。方法取新生24h的Wistar大鼠海马NSCs进行培养、鉴定。选7日龄Wistar大鼠制备HIBD动物模型,7d后行损伤侧侧脑室移植。将实验大鼠随机分为9组:正常组、模型组、磷酸盐缓冲液移植组、NSCs移植组、BDNF组、BDNF+NSCs移植组、NT-3组、NT-3+NSCs移植组、BDNF+NT-3+NSCs移植组。移植4周后进行功能实验,取脑组织进行免疫组化及免疫荧光检查。结果从新生大鼠海马可成功培养出NSCs,并表达NSCs的标志物神经巢蛋白(neuroepi-thelial stem cell protein,Nestin),NSCs可分化为神经元及星形胶质细胞。Y迷宫实验:NSCs移植组达到学会的次数(163±11.60)n,正确记忆次数(5.00±1.13)n;BDNF组(150.00±8.94,5.17±1.47)n;BDNF+NSCs移植组(111.25±13.56,6.23±1.60)n;NT-3组(153.56±10.35,5.07±1.03)n;NT-3+NSCs移植组(117.27±11.4,6.30±1.1)n;BDNF+NT-3+NSCs移植组(110±11.55,6.30±1.1)n。悬吊实验:NSC移植组平均(30.10±11.8)s;BDNF组(36.25±10.98)s;BDNF+NSCs移植组(43.6±10.56)s;NT-3组(34.95±10.15)s;NT-3+NSCs移植组(40.64±10.6)s;BDNF+NT-3+NSCs移植组(42.20±6.25)s。斜坡试验:NSCs移植组平均(20.3±8.25)s;BDNF组(14.33±2.88)s;BDNF+NSC移植组(11.17±2.48)s;NT-3组(15.26±2.98)s;NT-3+NSC移植组(12.8±3.65)s;BDNF+NT-3+NSCs移植组(12.9±5.18)s。说明各联合移植组大鼠学习记忆能力及神经功能与NSCs移植组比较,明显改善(P<0.05),而NSCs组大鼠学习记忆能力及神经功能与HIBD组比较,差异无统计学意义(P>0.05)。各联合移植组损伤侧皮层、海马存活的NSCs数量(BDNF+NSC移植组9.31±0.71个,10.10±1.65个;NT-3+NSCs移植组6.18±1.83个,8.18±3.06个;BDNF+NT-3+NSCs移植组为9.58±1.61个,11.45±1.36个)与NSCs组(3.33±0.24个,4.22±0.33个)比较明显增多(P<0.05),且NSCs分化为神经元的比例明显增多,与NSCs移植组比较,有统计学意义(P<0.05),但双因子联合移植组与其单因子移植组比较,差异无统计学意义(P>0.05)。结论BDNF、NT-3与NSCs联合移植是治疗HIBD的有效方法。
Objective To observe the therapeutic effect of neurotrophic factor combined with neural stem cells (NSCs) on hypoxic-ischemic brain damage (HIBD) rats. Methods The hippocampal NSCs of newborn Wistar rats were cultured and identified. The 7-day-old Wistar rats were selected to prepare the HIBD animal model and the injured lateral ventricle was implanted 7 days later. The experimental rats were randomly divided into 9 groups: normal group, model group, phosphate buffered saline transplantation group, NSCs transplantation group, BDNF group, BDNF + NSCs transplantation group, NT-3 group, NT-3 + NSCs transplantation group, BDNF + NT-3 + NSCs transplantation group. Four weeks after transplantation, functional experiments were performed, and brain tissue was harvested for immunohistochemistry and immunofluorescence. Results NSCs could be successfully cultured from the hippocampus of neonatal rats, and expressed as a neuroepithelial stem cell protein (NSN), which can differentiate into neurons and astrocytes. Y maze test: The number of learned NSCs transplantation group was (163 ± 11.60) n, the correct number of memory (5.00 ± 1.13) n; BDNF group (150.00 ± 8.94,5.17 ± 1.47) n; BDNF + NSCs transplantation group , 6.23 ± 1.60) n NT-3 group (153.56 ± 10.35,5.07 ± 1.03) n NT-3 + NSCs transplantation group (117.27 ± 11.4,6.30 ± 1.1) n and BDNF + NT-3 + NSCs transplantation group 110 ± 11.55, 6.30 ± 1.1) n. In the suspension group, the mean survival time of the NSC transplantation group was (30.10 ± 11.8) s, that of the BDNF group (36.25 ± 10.98 s), that of the BDNF + NSCs transplantation group (43.6 ± 10.56 s), NT-3 group + NSCs transplantation group (40.64 ± 10.6) s and BDNF + NT-3 + NSCs transplantation group (42.20 ± 6.25) s. In the slope test, the average number of NSCs transplantation group was (20.3 ± 8.25) s; BDNF group (14.33 ± 2.88) s; BDNF + NSC transplantation group (11.17 ± 2.48) s; NT- NSC transplantation group (12.8 ± 3.65) s and BDNF + NT-3 + NSCs transplantation group (12.9 ± 5.18) s. Compared with NSCs transplantation group, the learning and memory abilities and neurological functions of rats in allograft transplantation groups were significantly improved (P <0.05), while there was no significant difference in learning and memory ability and neurological function between NSCs group and HIBD group (P > 0.05). The number of NSCs surviving in the lateral cortex and hippocampus in each transplantation group was 9.31 ± 0.71 and 10.10 ± 1.65 in the group of BDNF + NSC transplantation and 6.18 ± 1.83 and 8.18 ± 3.06 in the group of NT-3 + NSCs transplantation respectively. BDNF + NT- (P <0.05). Compared with NSCs group (3.33 ± 0.24, 4.22 ± 0.33), the percentage of NSCs differentiated into neurons increased significantly (P <0.05) Compared with NSCs transplantation group, the difference was statistically significant (P <0.05), but there was no significant difference between the two factors combined transplantation group and the single factor transplantation group (P> 0.05). Conclusion Combined transplantation of BDNF, NT-3 and NSCs is an effective method for the treatment of HIBD.