本文選題：大鼠腦挫傷　切入點：骨髓間充質干細胞移植　出處：《昆明醫(yī)科大學》2013年碩士論文

【摘要】：目的：探討B(tài)MSC通過頸外動脈移植后對腦外傷大鼠神經功能和學習記憶的影響,為臨床應用BMSC治療TBI提供理論依據(jù)及新方法。 方法：選用健康清潔級Sprague-dawley(SD)大鼠10只,進行水迷宮測試后隨機分為TBI組和BMSC移植組。采用Freedom自由落體器制作大鼠皮質運動區(qū)腦挫傷模型,將培養(yǎng)純化并鑒定的BMSC于術后當天經頸外動脈移植入體內；術后1d、3d、7d、15d行改良神經功能缺失(NSS)評分,并于術后15天進行水迷宮測試以觀察動物學習記憶能力。15天時取腦組織用免疫熒光技術在熒光顯微鏡下檢測hochest標記的移植BMSC在體內存活、遷移情況。 結果：1.骨髓間充質干細胞體外培養(yǎng)及簽定本實驗最初分離的細胞呈懸浮狀態(tài),6h后即有細胞貼壁。為去除成纖維細胞,采用差速貼壁法于10h將全量換出液體,移入另一24孔板。24h后貼壁細胞增多,胞體逐步呈扁平的梭形、多角形。4d左右,原代細胞均勻于瓶底生長,細胞形態(tài)為長梭形,多角形,有不規(guī)則突起。傳至3-4代可用于移植。免疫組化染色顯示,BMSC呈CD44陽性染色呈棕黃色。 2.成功建立腦外傷大鼠動物模型 大鼠給予Freedom自由落體器撞擊傷后,大部分模型顱窗出血嚴重,行止血后,挫傷部位腦組織腫脹,膨出開顱窗口。行為學觀察顯示,單純手術組和干細胞移植組,大部分大鼠發(fā)生抽搐,癱瘓不能正常行走。NSS評分發(fā)現(xiàn),TBI大鼠四肢與軀干失去平衡功能,容易跌落。第1天和第3天單純手術組和細胞移植組各組大鼠NSS評分無明顯差異。而BMSC移植組的NSS評分至第7天后(6.5±1.19)與單純手術組(TBI)(8.75±0.68)比較顯著減少(P0.01),第15天也有統(tǒng)計學意義。 3.學習和記憶評價： 定位航行測試顯示：TBI術后與術前在Morris水迷宮逃避潛伏期相比,明顯延長。而BMSC移植組(20.48±2.29)與TBI組(85.93±47.48)比較,逃避潛伏期顯著縮短(P0.01)。空間探索結果顯示：撤去站臺后,BMSC移植組與TBI組比較,其在目標象限時間、路程百分比均顯著增高(P0.01),BMSC移植組穿越站臺次數(shù)也顯著增加(P0.01)。TBI組和術前組在目標象限時間、路程百分比和穿越站臺次數(shù)比較,均顯著降低(P0.01)。 4.熒光顯微鏡觀察BMSC移植入體內后分化、遷移：BMSC經頸外動脈移植入體內后在熒光顯微鏡下可見藍色細胞核,移植BMSC在皮質散在分布,整合于宿主腦組織。 結論：BMSC經頸外動脈移植入TBI大鼠體內,能有效存活、遷移并促進大鼠神經功能和學習記憶能力的恢復,表明BMSC經頸外動脈移植能夠對大鼠腦外傷產生治療效應。
[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.

【學位授予單位】：昆明醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R651.15

【參考文獻】

相關期刊論文 前4條

1 王公平;楊波;關方霞;杜英;常克亮;宋來君;胡祥;曾光偉;;不同途徑羊膜間充質干細胞移植治療大鼠腦外傷[J];中南大學學報(醫(yī)學版);2008年10期

2 趙鳳艷;屈藝;唐彬秩;毛萌;母得志;;新生大鼠缺氧缺血性腦損傷后細胞凋亡與端粒酶逆轉錄酶的表達[J];中國修復重建外科雜志;2010年05期

3 林建華;王長f;吳朝陽;;人腦源性神經營養(yǎng)因子基因修飾BMSCs經靜脈移植對大鼠損傷脊髓結構和功能影響的初步研究[J];中國修復重建外科雜志;2010年08期

4 趙富生;武庚;武楊;金秀東;張際緋;;雪旺細胞對不同年齡大鼠BMSCs分化的影響[J];中國修復重建外科雜志;2011年02期

，

本文編號：1668468