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目的研究神经营养素3(NT-3)及神经干细胞联合移植对缺氧缺血性脑损伤(HIBD)大鼠神经功能恢复及神经干细胞分化为神经元的比例的影响,并探讨其可能机制。方法取新出生的24 h 内的 wistar 大鼠,从海马中分离神经细胞,进行培养、鉴定。用出生7 d 的 wistar 新生大鼠制作缺氧缺血性脑损伤的动物模型,模型成功后7 d 进行移植。将实验大鼠随机分为5组:正常组、模型组、假移植组、神经干细胞移植组、NT-3及神经干细胞联合移植组(简称联合移植组),每组12只。移植部位为损伤同侧侧脑室。移植后4周进行功能实验,取脑组织进行免疫组化及免疫荧光检查。结果从新生鼠海马中成功培养出神经干细胞,培养条件下呈悬浮状态生长,形成神经球,绝大多数的细胞表达神经干细胞的标志物神经巢蛋白(nestin)。说明培养的细胞大部分为神经干细胞,细胞纯度达90%以上。Y 迷宫实验结果:神经干细胞移植组平均(163±11.6)次学会,记忆的平均正确率为(50±13.3)%;神经干细胞(NSC)+NT-3联合移植组平均(117.27±11.04)次学会,记忆的正确率平均(63±11.2)%。握持牵引实验结果:神经干细胞移植组平均(30.1±11.8)s,右下肢能放置的占40%;NSC+NT-3联合移植组平均(40.64±10.6)s,右下肢能放置的占72.73%。斜坡实验结果:神经干细胞移植组平均(20.3±8.25);NSC+NT-3联合移植组平均(12.9±5.15)s。说明接受 NT-3及神经干细胞移植组大鼠的学习能力、记忆能力及肢体功能与单纯神经干细胞移植组相比有明显提高,差异具有统计学意义(P<0.05)。其神经干细胞分化成神经元的比例(50%)与单纯神经干细胞移植组(30%)比有明显增多。结论 NT-3与神经干细胞联合移植能提高缺氧缺血性脑损伤大鼠的学习、记忆能力和肢体功能,并能提高神经干细胞向神经元转化的比率,NT-3和神经干细胞联合移植较单独神经干细胞移植对缺氧缺血性脑损伤有更好的治疗效果。
Objective To investigate the effects of NT-3 and neural stem cell transplantation on neurological function recovery and neural stem cell differentiation into neurons in hypoxic-ischemic brain damage (HIBD) rats and its possible mechanism. Methods Wistar rats within 24 h after birth were isolated from the hippocampus and cultured for identification. Animal model of hypoxic-ischemic brain damage was made with wistar neonatal rats of 7 days old. The model was transplanted 7 days after successful. The experimental rats were randomly divided into 5 groups: normal group, model group, sham transplanted group, neural stem cell transplantation group, NT-3 and neural stem cell transplantation group (referred to as combined transplantation group), 12 in each group. Transplant site for the damage ipsilateral lateral ventricle. Functional tests were performed 4 weeks after transplantation, and brain tissue was harvested for immunohistochemistry and immunofluorescence. Results Neural stem cells were successfully cultured in neonatal rat hippocampus and grew in suspension in culture condition to form neurospheres. Most of the cells expressed neural stem cell marker nestin. Explain that the majority of cultured cells are neural stem cells, with cell purity of over 90%. The results of Y maze test showed that the average correct rate of memory (+13 ± 11.6) times in neural stem cell transplantation group was (50 ± 13.3)% and that in NSC + NT-3 combined group was (117.27 ± 11.04) times Learning, the correct rate of memory average (63 ± 11.2)%. In the group of NSC + NT-3 combined transplantation, the mean (40.64 ± 10.6) s in the NSC transplantation group and 72.73% in the right lower limbs group (30.1 ± 11.8) s in the NSCs transplantation group and 40% in the right lower limbs group %. The results of slope experiment showed that the mean value of neural stem cell transplantation group was (20.3 ± 8.25) and that of NSC + NT-3 combined transplantation group was (12.9 ± 5.15) s. The results showed that the learning ability, memory ability and limb function of NT-3 and NSCs transplantation group were significantly higher than that of NSCs transplantation group (P <0.05). The proportion of NSCs differentiated into neurons (50%) was significantly higher than that of NSCs alone (30%). Conclusion Transplantation of NT-3 and neural stem cells can enhance the learning, memory and limb function of rats with hypoxic-ischemic brain damage and increase the ratio of neural stem cells to neurons. NT-3 and neural stem cell transplantation combined Single neural stem cell transplantation for hypoxic-ischemic brain injury have a better therapeutic effect.