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研究生:廖健凱
研究生(外文):Chien-Kai Liao
論文名稱:移植嗅鞘細胞與組織膠或加入神經生長因子三至第八胸髓截斷的大鼠中可有部分運動功能之恢復
論文名稱(外文):Transplantation of Olfactory Ensheathing Cells Combined with Fibrin Glue Mixed with/without Neurotrophin-3 in the T8-Transected Rat Spinal Cord Improves Functional Recovery
指導教授:傅毓秀傅毓秀引用關係鄭宏志鄭宏志引用關係
指導教授(外文):Yu-Show FuHenrich Cheng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:63
中文關鍵詞:脊髓截斷嗅鞘細胞嗅神經包被細胞神經生長因子三
外文關鍵詞:Spinal cordTransectionOlfactory ensheathing cells(OECs)Neurotropihn-3(NT-3)
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脊髓截斷會導致受損段以下的完全癱瘓和感覺喪失。於修復脊髓損傷的實驗中,植入嗅鞘細胞目前被認為是很有效的一種方式。目前已成功自成年大鼠嗅球中培養出表現GFAP,S100,P75NTR的嗅鞘細胞。
存在於脊髓皮質徑的神經生長因子三,被證實在脊髓傷害中能對損傷的軸突產生有效的再生作用。但嗅球培養出的嗅鞘細胞卻表現很低量的神經生長因子三。而我們的目標就是在檢驗於第八胸髓截斷的大鼠中,植入混有神經生長因子三及嗅鞘細胞的組織膠是否可促進其功能性的回復?嗅鞘細胞(十五萬)混入含有或不含有神經生長因子三(二微克)的組織膠中, 之後植入第八胸髓完全截斷的成年大鼠脊髓中。同時在斷端的前後一公厘處分別注入嗅鞘細胞(四萬)。包含開放場域行走測試(BBB) 及綜合行為分數(CBS)的後肢評估,在手術後每兩星期測試一次至手術後第十六週為止。發現嗅鞘細胞混入含有或不含有第三神經生長因子的兩群實驗組,在術後第十四週及第十六週時的綜合行為分數(CBS)相對於控制組(僅作脊髓全截斷手術)皆有顯著性的差異(P<0.05 ANOVA),在第十、十二、十六週時的開放場域行走測試(BBB),也有顯著性的差異(P<0.05 ANOVA)。
年大鼠於手術後第十六週,評估行為後犧牲,將脊髓組織處理後作冷凍切片,採免疫細胞染色法偵測截斷處是否有神經軸突的生長,以神經纖維(Neurofilament (NF)) 和GAP-43(新生軸突會表現的蛋白)為標記工具。發現控制組在第七胸髓的神經纖維(NF)標記數量比嗅鞘細胞混入含有神經生長因子三的實驗組來得少。而在截斷區域,控制組無法發現有神經纖維標記的細胞,但嗅鞘細胞混入含有神經生長因子三的實驗組則可發現。另外於截斷處,嗅鞘細胞混入含有神經生長因子三的實驗組可發現GAP-43的標記細胞,但在控制組中沒有發現。
綜合以上結果,發現(1)來自成年大鼠嗅球培養的細胞,可表現嗅鞘細胞已知的標記。(2)嗅鞘細胞組和嗅鞘細胞混入第三神經生長因子組,在第十二週(BBB) 第十四週(CBS),及第十六週(CBS. BBB)顯著的影響大鼠的後肢運動功能。 (3) 嗅鞘細胞混入神經生長因子三此組有益新生的神經軸突生長通過截斷區域。
Spinal cord transection results in permanent deficits involving complete paralysis and sensory loss below the level of lesion. Olfactory ensheathing cells (OECs) are regarded as the best therapeutic agent for use in the repair of spinal cord injury. We successfully established OEC with over 95% cells expressing GFAP, S100 and p75NGFR.
Neurotrophin-3 (NT-3), a putative neurotrophic factor in the rubro- and cortico-spinal tracts has been shown to effectively promote regeneration of damaged axons into the spinal cord. However, its expression is low in OEC. The present work therefore examines the effects of OEC transplant combined with fibrin glue mixed with NT3 on functional recovery after T8-transection.
OEC, prelabelled with 5-bromo-2-deoxyuridine or green fluorescence protein, were mixed with fibrin glue with/without NT-3 and transplanted into the transected spinal cord. Behavior tests (BBB and CBS) and were conducted every other week. By post operation , significant functional recovery in hind limb usage occurred in OECs or OECs + NT3 transplanted rats groups compared with control groups, transplanted with HBSS alone(p<0.05 ANOVA). However, none of the animals demonstrated coordinated fore and hind limb movement or the ability to bear weight on the hind limbs.
The immunhistochemistry showed that, the T7 level of OECs + NT3 groups presented more neurofilament and serotonin marker than control groups. At the transected site, neurofilament and serotonin marker can be found in the OECs + NT3 groups but not in the control groups.
In conclusion, our results demonstrated that (1) The primary cultured cells of OECs from olfactory bulb can present the GFAP, S100 and p75NGFR. (2) Fibrin glue enwrapped OECs with/without NT-3 transplanted rats showed significantly higher functional recovery in hind limb. (3) Fibrin glue enwrapped OECs with/without NT-3 transplanted rats facilitated the newly regenerated axon to pass the transected site.
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