Shootin1基因转染骨髓间充质干细胞移植治疗大鼠脊髓损伤的研究

发布时间：2018-05-04 15:10

本文选题：脊髓损伤 + 骨髓间充质干细胞　；参考：《南方医科大学》2017年硕士论文

【摘要】：背景:脊髓损伤(spinal cord injury,SCI)是生产生活中时常发生的一种后果比较严重的损伤。随着工业时代的飞速发展,交通事故、运动损伤和高处坠落等导致SCI患者数量逐年上升,且多发生于青壮年人群中。脊髓损伤后,神经元不能再生,常常容易发生截瘫,治疗难效果不理想,至今人们没有找到好的方法去解决这一难题。一般临床上采取手术减压,结合药物治疗。药物治疗方面用甲强龙冲击疗法,但是治疗效果不理想。因此,我们一直在探索应用一些新的技术手段提高SCI的疗效,使得脊髓损伤后损伤的轴突能再生,重建脊髓功能,尽快将之应用于临床治疗中。近年来随着组织工程技术研究的兴起以及对SCI后的病理改变、机理认识不断深入的研究,科学家们提出采用细胞移植,另外结合基因工程技术,使得脊髓损伤的治疗成为崭新的有效的方法变得可能。目的:探讨联合用腺病毒转染目的基因Shootin1的骨髓间充质干细胞移植治疗脊髓损伤的疗效,为进一步研究治疗脊髓损伤的方法作出有益的探索。方法:1.全骨髓贴壁培养法分离培养骨髓间充质干细胞,观察细胞形态,并进行流式细胞学鉴定。2.以腺病毒为载体,体外转染Shootin1进入BMSC,检测Shootin1蛋白的表达情况;3.在体外进行神经诱导分化,观察细胞是否向神经元样细胞分化;4.建立大鼠脊髓撞击损伤模型,在脊髓损伤后半小时内在原位分别注射移植Shootin1-BMSC和BMSC来进行脊髓损伤后修复治疗,每周对大鼠运动功能进行BBB评分,5周后处死大鼠,取脊髓标本进行冰冻切片,进行免疫荧光检测神经元相关标记蛋白。结果:1.同未转染Shootin1的骨髓间充质干细胞相比,携带Shootin1的慢病毒感染BMSCs能持续稳定高水平表达Shootin1蛋白;2.在体外经过神经诱导分化7天,Shootin1转染的BMSC与正常BMSC细胞形态上均发生了变化,我们通过IF检测神经前体细胞特异性标记Nestin和神经元特异性核蛋白NeuN的发现,Shootin1-BMSC和BMSC两组Nestin与NeuN蛋白的表达并无明显差异;3.骨髓间充质干细胞移植后,进行运动功能评分,发现细胞移植组与对照组相比,其运动功能恢复有明显的变化,并且Shootin1-BMSC移植组的BBB评分最高,但Shootin1-BMSC移植组与BMSC组的评分相比(P0.05)没有统计学意义。结论:1.本实验证明了 BMSCs细胞移植可以有效地促进脊髓损伤后运动功能恢复;2.Shootin1蛋白可以促进脊髓损伤后损伤区域神经再生和功能恢复,但其作用较BMSCs直接移植组并不明显。
[Abstract]:Background: spinal cord injury (sci) is a serious injury in life. With the rapid development of the industrial age, traffic accidents, sports injuries and falling from height lead to an increase in the number of SCI patients, and most of them occur among the young and middle-aged people. After spinal cord injury, the neurons can not regenerate, and are prone to paraplegia. So far, people have not found a good way to solve this problem. General clinical use of surgical decompression, combined with drug treatment. The medicine treatment aspect uses the nail strong dragon pulse therapy, but the treatment effect is not ideal. Therefore, we have been exploring some new techniques to improve the efficacy of SCI, so that the injured axons can regenerate after spinal cord injury, reconstruct spinal cord function, and apply it to clinical treatment as soon as possible. In recent years, with the rise of tissue engineering technology and the pathological changes after SCI, the understanding of mechanism has been deeply studied. Scientists have proposed the use of cell transplantation, in addition to the combination of genetic engineering technology. This makes the treatment of spinal cord injury a new and effective method. Objective: to investigate the effect of bone marrow mesenchymal stem cell transplantation combined with adenovirus transfected target gene Shootin1 in the treatment of spinal cord injury (sci). Method 1: 1. Bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherent culture method. Using adenovirus as vector, Shootin1 was transfected into BMSC in vitro to detect the expression of Shootin1 protein. Neural differentiation was conducted in vitro to observe whether the cells were differentiated into neuron-like cells. The spinal cord impingement injury model was established in rats. Shootin1-BMSC and BMSC were injected into the spinal cord injury within half an hour after spinal cord injury to repair the spinal cord injury. The rats were killed 5 weeks later with the BBB score of motor function every week. The spinal cord specimens were taken for frozen section and immunofluorescence was used to detect the neuron-related marker protein. The result is 1: 1. Compared with untransfected bone marrow mesenchymal stem cells (BMSCs), lentivirus infected with Shootin1 could maintain high and stable expression of Shootin1 protein in BMSCs. The morphologic changes of BMSC transfected with Shootin1 and normal BMSC cells were observed on the 7th day after neuronal induction and differentiation in vitro. We found that there was no significant difference in the expression of Nestin and NeuN protein between Shootin 1-BMSC and BMSC groups by if detection of neuronal specific Nestin and neuron-specific nucleoprotein NeuN. After bone marrow mesenchymal stem cell transplantation, the motor function score was evaluated. It was found that the motor function recovery of the cell transplantation group was significantly different from that of the control group, and the BBB score of the Shootin1-BMSC transplantation group was the highest. However, the score of Shootin1-BMSC transplantation group was not significantly higher than that of BMSC group (P 0.05). Conclusion 1. This study demonstrated that BMSCs cell transplantation can effectively promote motor function recovery after spinal cord injury. 2. Shootin1 protein can promote nerve regeneration and functional recovery in injured area after spinal cord injury, but its effect is not obvious compared with BMSCs direct transplantation group.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

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