颈外动脉BMSC移植对脑外伤大鼠神经功能和学习记忆的影响

发布时间：2018-03-26 15:35

本文选题：大鼠脑挫伤　切入点：骨髓间充质干细胞移植　出处：《昆明医科大学》2013年硕士论文

【摘要】：目的：探讨BMSC通过颈外动脉移植后对脑外伤大鼠神经功能和学习记忆的影响,为临床应用BMSC治疗TBI提供理论依据及新方法。方法：选用健康清洁级Sprague-dawley(SD)大鼠10只,进行水迷宫测试后随机分为TBI组和BMSC移植组。采用Freedom自由落体器制作大鼠皮质运动区脑挫伤模型,将培养纯化并鉴定的BMSC于术后当天经颈外动脉移植入体内；术后1d、3d、7d、15d行改良神经功能缺失(NSS)评分,并于术后15天进行水迷宫测试以观察动物学习记忆能力。15天时取脑组织用免疫荧光技术在荧光显微镜下检测hochest标记的移植BMSC在体内存活、迁移情况。结果：1.骨髓间充质干细胞体外培养及签定本实验最初分离的细胞呈悬浮状态,6h后即有细胞贴壁。为去除成纤维细胞,采用差速贴壁法于10h将全量换出液体,移入另一24孔板。24h后贴壁细胞增多,胞体逐步呈扁平的梭形、多角形。4d左右,原代细胞均匀于瓶底生长,细胞形态为长梭形,多角形,有不规则突起。传至3-4代可用于移植。免疫组化染色显示,BMSC呈CD44阳性染色呈棕黄色。 2.成功建立脑外伤大鼠动物模型大鼠给予Freedom自由落体器撞击伤后,大部分模型颅窗出血严重,行止血后,挫伤部位脑组织肿胀,膨出开颅窗口。行为学观察显示,单纯手术组和干细胞移植组,大部分大鼠发生抽搐,瘫痪不能正常行走。NSS评分发现,TBI大鼠四肢与躯干失去平衡功能,容易跌落。第1天和第3天单纯手术组和细胞移植组各组大鼠NSS评分无明显差异。而BMSC移植组的NSS评分至第7天后(6.5±1.19)与单纯手术组(TBI)(8.75±0.68)比较显著减少(P0.01),第15天也有统计学意义。 3.学习和记忆评价：定位航行测试显示：TBI术后与术前在Morris水迷宫逃避潜伏期相比,明显延长。而BMSC移植组(20.48±2.29)与TBI组(85.93±47.48)比较,逃避潜伏期显著缩短(P0.01)。空间探索结果显示：撤去站台后,BMSC移植组与TBI组比较,其在目标象限时间、路程百分比均显著增高(P0.01),BMSC移植组穿越站台次数也显著增加(P0.01)。TBI组和术前组在目标象限时间、路程百分比和穿越站台次数比较,均显著降低(P0.01)。 4.荧光显微镜观察BMSC移植入体内后分化、迁移：BMSC经颈外动脉移植入体内后在荧光显微镜下可见蓝色细胞核,移植BMSC在皮质散在分布,整合于宿主脑组织。结论：BMSC经颈外动脉移植入TBI大鼠体内,能有效存活、迁移并促进大鼠神经功能和学习记忆能力的恢复,表明BMSC经颈外动脉移植能够对大鼠脑外伤产生治疗效应。
[Abstract]:Objective: To explore the effect of BMSC on neurological function and learning and memory after traumatic brain injury in rats, and provide a theoretical basis and a new method for the treatment of TBI with BMSC.
Methods: healthy clean Sprague-dawley (SD) 10 rats by water maze test were randomly divided into TBI group and BMSC group. The production of motor cortex brain contusion model in rats with Freedom free fall, will develop the purification and identification of BMSC on the day after the surgery via external carotid artery transplanted; after 1D, 3D, 7d, 15d improved neurological deficit score (NSS), and in 15 days after Morris water maze test was performed to observe the animal learning and memory ability in.15 days from brain tissue detection of hochest labeled BMSC under fluorescent microscope by immunofluorescence migration in vivo,.
Results: 1. bone marrow mesenchymal stem cells cultured in vitro and the signing of this initial experiment of isolated cells was suspended and 6h after removal of adherent cells. Fibroblasts, using the differential adherence method in 10h will be a full amount for a liquid, increase of adherent cells into another 24 hole plate.24h the cell body, gradually spindle shaped flat and polygonal.4d, primary cell growth even at the bottom of the bottle, cells were fusiform, polygonal, irregular protrusions. Spread to the 3-4 generation can be used for transplantation. The immunohistochemical staining showed BMSC positive staining of CD44 was brownish yellow.
2. successful establishment of rat model of brain injury
Rats were treated with Freedom for free fall injuries, most models of cranial window severe bleeding, with blood after brain contusion, swelling, swelling behavior observation window. Craniotomy, surgery group and stem cell transplantation group, most seizures in rats, paralysis can not walk properly.NSS score, limbs and trunk big TBI were out of balance function, easy to fall. No significant difference between the first and third day surgery group and cell transplantation group rats. NSS score and BMSC transplantation group NSS score to seventh days (6.5 + 1.19) and surgery group (TBI) (8.75 + 0.68) is significantly reduced (P0.01), there was also statistical significance for fifteenth days.
3. learning and memory evaluation:
The navigation test showed that TBI patients before and after surgery in the Morris water maze escape latency was significantly prolonged compared. BMSC transplantation group (20.48 + 2.29) and TBI group (85.93 + 47.48), escape latency was significantly shortened (P0.01). The results showed that space exploration from the site after the BMSC transplantation group compared with the TBI the group in the target quadrant time distance percentage were significantly increased (P0.01), BMSC transplantation group was also significantly increased the number of cross platform (P0.01) of.TBI group and the preoperative group in the target quadrant time comparison distancepercentage and the times to cross the target, was significantly decreased (P0.01).
BMSC transplanted after differentiation, migration was observed in 4. fluorescent microscopy: BMSC via external carotid artery transplantation in vivo under fluorescent microscope visible blue nuclei, the transplantation of BMSC in the cortex were distributed, integrated in the host brain tissue.
Conclusion: BMSC can effectively survive, migrate and promote the recovery of neural function and learning and memory ability in TBI rats after external carotid artery transplantation. It indicates that BMSC can exert a therapeutic effect on traumatic brain injury in rats through external carotid artery transplantation.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R651.15

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