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9d后的细胞,调整细胞终浓度至5×10^10L^-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10min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,2μL/点,细胞总数约4×10^5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15d和30d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30min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,2d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15d,30d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30d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。
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.（1）Thirty-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.（2）After 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 5×10^10 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 4×10^5 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.（3）The hindlimb locomotor function of rats in three groups were detected with Tadov 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 μ L 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.（1）Detection 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）.（2）Inclined plane test： The identical results were observed in the hindlimb locomotor function detection.（3）HRP 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.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第12期2263-2266,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(3000172)
广西省科学研究与技术开发基金(2005)~~
