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研究生:黃繼霆
研究生(外文):Chi-ting Huang
論文名稱:植入基因轉殖之嗅神經包被細胞可促進脊髓損傷後之神經再生
論文名稱(外文):Gene Transfer and Transplantation of OECs promote axonal regeneration in transected rat spinal cord
指導教授:黃勇三鄭宏志鄭宏志引用關係
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:67
中文關鍵詞:脊髓損傷嗅神經包被細胞神經再生
外文關鍵詞:spinal cord injuryolfactory ensheathing cellsaxonal regeneration
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嗅神經包被細胞(olfactory ensheathing cells, OECs)是一種特別的神經膠細胞,同時具有許旺氏細胞及星形膠細胞的特性。數篇文獻曾證實脊髓損傷後植入OECs能促進脊髓神經軸突的生長。為增進OECs協助神經再生的功能,以重組腺病毒載體將合成前列腺素I2 (Prostacyclin , PGI2)的酵素送入OECs內。PGI2經由cyclooxygenase (COX-1 or COX-2)及PGI2 synthase (prostacyclin synthase, PGIS)將花生四烯酸(arachidonic acid, AA)轉化成PGI2。PGI2具有血管擴張、抑制血小板凝集及細胞保護作用。
OECs從成年大白鼠的嗅球中分離培養,培養出的OECs,百分之95以上具有p75NTR、S-100之細胞免疫標記特性。以重組腺病毒載體帶綠螢光蛋白(green fluorescent protein, GFP)基因轉殖後之OECs其細胞免疫標記特性並不會改變且顯示高感染性。
培養出的OECs本身具有較高COX-2及COX-1蛋白質量,並且有大量的PGE2產生,PGIS蛋白質量則較低。因此,以PGIS基因轉殖之重組腺病毒載體讓OECs表現外來基因以產生PGI2。之後以大白鼠第八胸髓完全截斷之動物脊髓損傷模式,於切斷區(5~6mm gap)植入以組織膠(fibrin glue)包埋之OECs或基因轉殖OECs。手術後一週,表現GFP的rAd-GFP-OECs可以在移植區內及移植區邊緣染到,但並未遷移於脊髓組織內。實驗動物於手術後第二週開始每隔一週作行為測試(BBB及CBS scores)。手術後十二週,以組織膠包埋rAd-PGIS-OECs植入脊髓完全截斷之大白鼠可以明顯觀察到其後肢行為恢復程度較移植OECs組好(P<0.05)。但並未發現有任何實驗動物有後肢與前肢的協調性,後肢也未能支撐體重。WGA-HRP順向神經追蹤試驗顯示只有在移植rAd-PGIS-OECs組中2隻實驗動物(40﹪)可在第十二胸髓節找到WGA-HRP標定的軸突。serotonin免疫組織化學染色顯示,移植OECs實驗組與移植rAd-PGIS-OECs實驗組中各有一隻 (16.7﹪)與三隻(60﹪)實驗動物,可在手術12週之後於橫切處後端的脊髓中發現5-HT positive軸突。
由以上結果可得知OECs包埋在組織膠中可存活,並可協助再生軸突成功的通過脊髓損傷部位。以組織膠包埋重組腺病毒載體PGIS基因轉殖之OECs移植於損傷的脊髓,無論在後肢功能的恢復上、神經軸突的再生上,皆較組織膠包埋OECs移植組好。
Olfactory ensheathing cells (OECs) are specialized glia that ensheathe bundles of olfactory axons. Having both Schwann cell-like and astrocyte-like characteristic, OECs have been reported as favorable substrate for axonal regeneration. Recent evidence suggests that OECs transplanted into injured spinal cord may facilitate axonal regeneration. In an attempt to improve the growth-promoting properties of OECs, the possibility of ex vivo adenoviral gene transfer of prostacyclin (PGI2) synthetic enzymes to OECs was examined. PGI2 is a potent inhibitor of platelet aggregation and a vasodilator. It has been demonstrated as cytoprotective agents against various stresses. Its biosynthesis is catalyzed by sequential actions of cyclooxygenase (COX-1 or COX-2) and prostacyclin synthase (PGIS) from arachidonic acid.
OECs were purified from adult rat olfactory bulb by their unique seeding properties to plastic dish. These cells were then expanded by DMEM/F12-based medium conditioned by astrocytes. Immunolabelling of these cells demonstrated that >95% of the cells expressed both S100 and p75, the low affinity nerve growth receptor. Infection of OECs with recombinant adenovirus (rAd) encoding green fluorescence protein (GFP) demonstrated highly efficient transfer. At 2 days after infection, almost all transduced OECs expressed high levels of green fluorescence without changes of their cellular phenotype.
Cultured OECs were found to express high levels of COX-1 and COX-2, concurrent with high production of PGE2, a pro-inflammatory agent. By contrast, the PGIS expression level is subtle. rAd-PGIS-infected OECs selectively augmented PGI2 production. Therefore, ex vivo rAd-GFP-(as vector control) or rAd-PGIS-transduced OECs were chosen for cell transplantation to T8-transected spinal cord. Suspension of rAd-GFP or rAd-PGIS-transduced OECs were mixed with fibrin glue before grafting into the transected (5~6mm gap) thoracic spinal cord. One week after surgery, GFP expressed OECs were detected within transected region but not inside the host cords. Beginning two weeks after surgery, behavior tests (BBB and CBS scores) were conducted every other week. By 12 weeks post-surgery, significant functional recovery in hind limb usage occurred in OECs or rAd-PGIS-infected OECs transplanted rats compared with controls, transplanted with culture medium alone. rAd-PGIS-OECs transplanted rats showed significantly higher BBB score than that of OECs-transplanted rats (P<0.05). However, none of the animals demonstrated coordinated fore and hind limb movements or the ability to bear weight on the hind limbs. Anterograde tracing by WGA-HRP from sensory motor cortex revealed that WGA-HRP labeled corticospinal tract fibers in the distal cord were found in two of rAd-PGIS-OECs transplanted rats (40﹪) at thoracic level 12. Furthermore, positive immunostaining of serotonergic axons in the distal cord were observed in one of OECs-transplanted rats (16.7﹪) and three of rAd-PGIS-OECs transplanted rats (60﹪).
In conclusion, our results demonstrated that (1) OECs mixed with fibrin glue survived and promoted regenerating nerve fibers traverse the lesion; (2) fibrin glue enwrapped rAd-PGIS-OECs transplanted rats showed significantly higher functional recovery in hind limbs and axonal regeneration than that of OECs transplanted rats.
中文摘要 I
英文摘要Abstract III
縮寫表 V
表次 VI
圖次 VII
第一章 緒言 1
第二章 文獻探討 3
第一節 脊髓損傷 3
2-1.1 脊髓之解剖構造 3
2-1.2 脊髓損傷之流行病學 3
2-1.3 脊髓損傷之致病機轉 4
第二節 脊髓損傷之實驗動物模式 5
第三節 脊髓損傷之實驗治療進展 7
第四節 嗅神經包被細胞 9
2-4.1 OECs之型態功能特性 9
2-4.2 OECs之細胞免疫特性 9
2-4.3 OECs對脊髓損傷的治療作用 10
第五節 重組腺病毒載體 11
第六節 前列腺素I2 12
第三章 材料與方法 15
第一節 實驗動物 15
第二節 嗅神經包被細胞(OECs)之培養 15
第三節 OECs之細胞特性鑑定 16
3-3.1 Hoechst標定 16
3-3.2 免疫螢光染色 16
第四節 重組腺病毒(rAd)感染OECs 17
第五節 OECs感染rAd後之細胞特性鑑定 17
第六節 OECs感染rAd後之轉殖基因表現測定 18
3-6.1 西方墨點法 18
3-6.2 高效液相層析法 20
第七節 動物脊髓損傷手術與OECs之移植 21
第八節 行為測試及評估 22
3-8.1 綜合行為評分表 22
3-8.2 BBB Score 22
第九節 WGA-HRP的追蹤手術 23
3-9.1 順向WGA-HRP的追蹤手術 23
3-9.2 逆向WGA-HRP的追蹤手術 23
第十節 實驗動物犧牲、灌流固定與切片 23
3-10.1 WGA-HRP追蹤手術之實驗動物 23
3-10.2 未經WGA-HRP追蹤手術之實驗動物 24
第十一節 WGA-HRP的呈色 25
第十二節 免疫組織化學染色 25
第十三節 統計分析 26
第四章 結果 27
第一節 OECs 培養與鑑定 27
第二節 OECs感染rAd後之細胞特性不變 27
第三節 OECs感染rAd後轉殖基因之表現 28
第四節 行為測試 28
第五節 神經軸突的再生 29
第六節 移植區內組織膠包埋GFP基因轉殖OECs的分佈情形 30
第五章 討論 50
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