课题背景：本课题前期受到国家自然科学基金资助资助，项目编号3000172，已经顺利结题。该课题应用研究得到解放军第三军医大学基础医学部组织学与胚胎学教研室与解放军第一八一医院皮肤科联合申请的广西省科学研究与技术开发基金资助，项目经费15万元，拟培养人胚胎干细胞，并着重其移植治疗临床相关病例的应用研究。

应用要点：①实验应用NT-3基因转染胚胎干细胞，以经典维甲酸4-/4+法体外诱导转染后的胚胎干细胞分化为神经前体细胞（神经元前体和胶质细胞前体），找到最佳的体外分化的条件。②将分化后的混合细胞移植到大鼠脊髓横断模型的损伤部位，观察移植物能否增强神经元功能的恢复。

偏倚或不足：由于实验周期限制，在脊髓损伤的移植治疗过程中存在一些观察盲区，如细胞移植后缺乏进行整个损伤神经元重建的研究，仅对损伤区域进行形态学观察难以说明神经元整体的恢复；另外NT3表达的研究存在一些不足。拟进行损伤区域近侧脊髓的连续切片，免疫细胞化学染色对神经元形态的恢复及NT3在蛋白水平的表达进行观察，经过三维重建进一步了解整个受损伤神经元的形态结构变化。

摘要
目的：由胚胎干细胞分化的神经细胞移植能够在一定程度上恢复脊髓损伤模型动物的功能，此发现使胚胎干细胞成为一种用于移植学研究的重要工具。观察经NT3基因转染修饰的小鼠MESPU35（ES-NT3）胚胎干细胞株移植对脊髓损伤动物脊髓结构和功能重建的恢复效果。
方法：实验于2004-09/12 在解放军第三军医大学组织胚胎学教研室实验室完成。①材料：清洁级成年Wistar大鼠36只，随机数字表法分为模型对照组、未转染细胞移植组、基因转染细胞移植组，12只/组，实验过程中对动物的处置符合动物伦理学标准。移植所用MESPU35细胞株和ES-NT3细胞株均由本室冻存。②实验方法：复苏MESPU35和ES-NT3细胞株，采用经典维甲酸4-/4+法诱导两种胚胎干细胞神经定向分化，收集分化9 d后的细胞，调整细胞终浓度至5×1010 L－1备用。各组大鼠均建立L4 脊髓损伤模型，在脊髓完全横断后10 min内，基因转染细胞移植组分多点缓慢注入ES-NT3胚胎干细胞悬液， 2 μL/点，细胞总数约4×105个，注射位置为损伤区域白质与灰质交界处；未转染细胞移植组同法注射MESPU35胚胎干细胞悬液，模型对照组注射等量生理盐水。③实验评估：各组大鼠分别于术前、术后即刻、术后15 d和30 d进行后肢运动功能Tarlov评分检测，分数越低表示运动功能恢复越差。后肢运动功能检测30 min后进行斜板实验，检测大鼠抓握和维持姿势能力。微型注射器将25％的辣根过氧化物酶2μL注入大鼠坐骨神经内，2 d后取L1~L4脊髓常规冰冻切片，参照Mesulam法进行过氧化物酶逆行追踪。
结果：因细菌感染，模型对照组、未转染细胞移植组、基因转染细胞移植组各死亡2只、1只、2只。①后肢运动功能检测：各组大鼠术后即刻后肢运动功能评分为0。术后15 d，30 d与模型对照组比较，未转染细胞移植组后肢运动功能评分升高(P < 0.01)，但未达到正常水平；基因转染细胞移植组恢复程度最高，后肢运动功能评分基本接近正常水平，明显高于未转染细胞移植组(P < 0.05)。②斜板试验：与后肢运动功能检测结果基本相似。③辣根过氧化物酶逆行追踪情况：模型对照组未见阳性神经元。术后30 d基因转染细胞移植组阳性神经元增至最多，脊髓结构恢复情况优于未转染细胞移植组。
结论：经NT3基因转染修饰的鼠MESPU35胚胎干细胞向神经定向诱导分化后，移植治疗脊髓损伤大鼠能更好地促进脊髓功能恢复和结构重建。
关键词：胚胎干细胞；NT-3基因；脊髓损伤；大鼠

秦茂林，杨卫兵，李红丽，李成仁，刘建军.小鼠胚胎干细胞NT3基因转染后移植对脊髓损伤的恢复作用[J].中国组织工程研究与临床康复，2008，12(12):2263-2266
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2263(ps).pdf]

Effects of neurotrophin-3 modified embryonic stem cells transplantation on the recovery of spinal cord injury

Abstract

AIM：Embryonic stem cell (ES)-derived neural cells transplantation can recover the function of spinal cord injury animal models, therefore ES cells plays an important role in transplantation researches. This study was designed to observe effects of mouse MESPU35 (ES-NT3 cells) modified by neurotrophin-3 gene (NT-3) on the structure and function recovery in rats with spinal cord injury.

METHODS: This experiment was conducted in laboratory of Department of Histology and Embryology, the Third Military Medical University of Chinese PLA from September to December in 2004.①Thirty-six clean-level adult Wistar rats were divided into three groups by the method of random digits table: model control group, transplantation control group and transplantation group, with 12 rats in each group. All the disposals were in accordance with the guideline of animal ethics. MESPU35 cell strain and ES-NT3 cell strain used for transplant were frozen in this laboratory.②After anesthesia, MESPU35 cell strain and ES-NT3 cell strain were induced to differentiate with typical retinoic acid 4-/4+ method. Nine days later, cells were collected and adjusted into a density of 5×1010 L-1. Rat spinal cord was exposed, and the transection model of injury was fabricated on L4 spine cord within 10 minutes. For the transplantation control group and transplantation group, the suspenseful fluid of MESPU35 and ES-NT3 cell strains (totally 4×105 cells), was slowly injected at the juncture between white matter and grey matter in lesioned spinal cord respectively, 2 μL for each lesion site. The rats of model control group were injected with the same dose of physiologic saline.③The hindlimb locomotor function of rats in three groups were detected with Tarlov score before operation, and at 0, 15, 30 days after operation. The lower score indicated poorer recovery of the hindlimb locomotor function. Thirty minutes after the detection, the inclined plane trial was performed to test the grasping and posture maintenance ability of the rats. Before drawing the materials from rats, 2 μL 25% horseradish peroxidase (HRP) was injected in sciatic nerve by microinjector. Two days later, L1–4 spinal cord sections of rats were frozen with routine method, and staining was performed according to Mesulam’s method to observe HRP retrogradation tracing.

RESULTS: Due to bacterial infection, there were 2, 1, 2 animals died respectively in model control group, transplantation control group and transplantation group.①Detection of hindlimb locomotor function: All rats were scored as 0 at 0 day after operation. Compared with the model control group, scores of hindlimb locomotor function at 15 and 30 days after operation increased obviously in the transplantation control group (P < 0.01), however still lower than normal level. Rats of transplantation group had the highest scores and were near to normal level, which were significantly higher than that of transplantation control group (P < 0.05).②Inclined plane test: The identical results were observed in the hindlimb locomotor function detection.③HRP retrogradation tracing test: No positive neurons were observed in model control group. The numbers of staining positive neuron increased to the most level in spinal cord of rats in transplantation group at 30 days after operation, and the structure of spinal cord had better recovery than transplantation control group.

CONCLUSION: Neural differentiation of rat MESPU35 ES cells modified by NT-3 is transplanted in rats with spinal cord injury, which can promote both function recovery and structure reconstruction of injured spinal cord.

Qin ML, Yang WB, Li HL, Li CR, Liu JJ.Effects of neurotrophin-3 modified embryonic stem cells transplantation on the recovery of spinal cord injury.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(12):2263-2266(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-12/12k-2263(ps).pdf]