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研究生:陳宏盈
論文名稱:以脈衝磁場刺激加上胚胎脊髓組織移植促進後肢癱瘓大鼠之脊髓再生
論文名稱(外文):Pulsed Electromagnetic Field plus Fetal Spinal cord Transplants Enhance the Regeneration of Spinal Cord in Paraplegic Rat
指導教授:黃勇三
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:102
中文關鍵詞:脊髓再生脈衝電磁場胚胎脊髓組織移植
外文關鍵詞:spinal cord regenerationpulsed electromagnetic fieldfetal spinal cordtransplantation
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本實驗以第八胸脊髓完全切斷之Spraque-Dawley雌鼠作為脊髓損傷的研究模式,一方面探討脈衝電磁場刺激能否促進脊髓再生反應,另一方面則探討胚胎脊髓組織移植加上脈衝電磁場刺激,在促進脊髓再生的效果上是否會有更好的成效。實驗分組中以手術後不作其他處理為控制組(Tx);術後隔日給予25 Gauss (2.5mT)、60 Hz的脈衝電磁場刺激、每日連續刺激6小時為電磁場刺激實驗組(PEMF);切斷脊髓後馬上移植懷孕13-14天的胚胎脊髓組織,為移植實驗組(Tp);手術後移植胚胎脊髓組織隔日以相同條件的電磁場刺激,為移植加上電磁場刺激的實驗組(Tp/PEMF)。沒有進行手術與其他處理的大鼠則作為正常組別(Intact)。
各組實驗動物分別於手術後第四、十二天犧牲,進行免疫化學染色、觀察並分別計算紅核(red nucleus)內表現c-Jun蛋白與βШ-tubulin之神經元數目,以評估脊髓受損後上位神經元短期內的存活與再生能力。於術後第三個月,各組大鼠分別在大腦運動皮質區、中腦紅核以及T12胸脊髓打入 Fluorogold,來標示手術區域的運動皮質脊髓徑軸突、紅核脊髓徑軸突生長的情形、以及標示神經軸突受損後有成功再生之上位神經元。並藉由觀察含serotonin (5-HT)之神經軸突在手術區域及其後側脊髓的生長情況,作為各組脊髓再生情形比較的依據。最後在手術後第一、二、三、四個月分別進行步態分析,評估各組大鼠後肢運動功能恢復差異。
實驗結果發現:在移植加上電磁場刺激之後,紅核內表現c-Jun蛋白與βШ-tubulin的神經元數量分別會增加2.4與1.1倍,其效果會超過單獨電磁場刺激(分別只有增加1.4倍與0.4倍)。利用神經軸突追蹤方式,在移植加上電磁場刺激組,位於中腦紅核,與髓腦的網狀結構區、縫核、前庭核均有被Fuorogold標定的神經元。在手術移植區域以及後端(T11)脊髓,均有serotonin axon生長,而corticospinal axons與rubrospinal axons 同樣可以生長到達T11胸脊髓。而電磁場刺激組,位於中腦紅核與髓腦網狀結構區也有被Fuorogold標定的神經元;在手術區域、與後方脊髓(T11)均有serotonin axons軸突生長;在T11胸脊髓則只有rubrospinal axons生長。經步態分析,在手術後3至4個月可見移植加上電磁場刺激組以及電磁場刺激組之大鼠其前進速度有明顯增加、且後肢有負重的腳步則分別恢復至正常大鼠的30%與33%,兩組之間並沒有明顯差異。
結論:在切斷T8胸脊髓後,給予電磁場刺激加上移植胚胎脊髓組織可以幫助脊髓上位神經元再生,有利於脊髓的修復,並幫助後肢運動功能的部份恢復。
Female Spraque-Dawley Rats were operated with T8 transection in these experiments. Surgical controls (Tx) were T8 transected without any other treatments. PEMF rats were transected with pulsed electromagnetic field stimulation (strength 25Gauss with frequency 60 Hz). Tp rats were transected with fetal spinal cord transplantation (embryonic 13~14 days). Tp/PEMF rats were transected with Tp plus PEMF and Intact rats were normal controls without T8 transection.
We evaluated the survival and regenerative capacity of injured neurons by comparing the c-Jun protein and βШ-tubulin expression in red nucleus of experimental rats 4 or 12 days after surgery. Neuroanatomical anterograde and retrograde tracing techniques were used to label the growth of corticospinal axons, rubrospinal axons in T8 lesion and the caudal spinal cord (T11), and to label the axontomized supraspinal neurons which regenerated their axon into the T12 spinal cord. Antibody against serotonin (5-HT) was used to visualize the serotonin axons growth pattern within the lesion site, transplant area and caudal spinal cord. Gait analysis was carried out 1, 2, 3, 4 month(s) after surgery and once in normal rats to evaluate the functional recovery of hindlimb movement.
Our data showed that application of Tp/PEMF could up-regulate the expression of c-Jun protein and βШ-tubulin in axotomized red nucleus neurons (2.4 folds increasing in c-Jun and 1.1 folds in βШ-tubulin), better than PEMF ( 1.4 folds in c-Jun and 0.4 fold in βШ-tubulin). The serotonin axons, corticospinal axons and rubrospinal axons in Tp/PEMF rats were able to growth through T8 lesion and back into caudal spinal cord (T11). By retrograde tracing, many neurons were Fluorogold labeled in several brainstem nuclei (including red n.、reticular formation、vestibular n. and raphe n.) in Tp/PEMF rats. But in PEMF rats, the Fluorogold labeled neurons were only seen in red n. and reticular formation. The serotonin axons and rubrospinal axons were able to growth through the T8 lesion and back into caudal spinal cord (T11). In gait analysis 3 month after surgery, Tp/PEMF and PEMF rats were faster than controls (Tx) by using their hindlimbs weight-bearing for 30% and 33% of the movements. There was no significant difference between Tp/PEMF and PEMF in hindlimb functional recovery.
In conclusion, pulsed electromagnetic field stimulation plus fetal spinal cord transplants can enhance the regenerative capacity of axotomized supraspinal neurons, promote spinal axons regeneration and partial functional recovery of hindlimb movement after T8 transection.
致謝………………………………………………………………………..Ⅰ
中文摘要……………………………………………………………..........Ⅱ
英文摘要Abstract…………………………………………………………Ⅳ
圖次………………………………………………………………………..Ⅵ
英文縮寫與中文對照……………………………………………………..Ⅷ
第一章 緒言………………………………………………………………..1
第二章 文獻探討…………………………………………………………..3
第一節 電磁場……………………………………………………...3
一、電磁場與人體健康……………………………………3
二、電磁場可能的作用機制………………………………5
三、電磁場與神經系統……………………………………9
第二節 胚胎脊髓組織移植……………………………………….12
第三節 神經元存活與再生能力………………………………….14
一、c-Jun蛋白表現………………………………………14
二、βⅢ tubulin表現………………………………………18
第三章 材料與方法………………………………………………....……21
一、實驗動物……………………………………………………21
二、顯微手術……………………………………………………21
三、脈衝電磁場刺激……………………………………………25
四、脊髓上位神經元再生能力之評估…………………………27
五、脊髓軸突再生反應之評估………………………………….29
六、後肢運動功能恢復之評估…………………………………33
七、數據分析……………………………………………………36
第四章 實驗結果…………………………………………………………37
一、脊髓上位神經元再生能力之評估……….……………………37
二、脊髓軸突再生能力的評估……………………………………40
三、後肢運動功能的恢復…………………………………………42
第五章 討論……………………………………………………...……….75
參考文獻………………………………………………………..…………89
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