【作者】 纪家武；

【导师】 王玮；

【作者基本信息】 福建医科大学 ， 人体解剖与组织胚胎学， 2005， 硕士

【摘要】 目的:通过观察大鼠行为学的改变及脊髓损伤(spinal cord injury,SCI)后神经细胞凋亡的情况,了解神经干细胞(neural stem cells,NSCs)移植对损伤脊髓功能修复的作用和对SCI大鼠神经细胞凋亡的影响,为临床治疗提供实验依据。 方法:①脊髓NSCs的培养和鉴定由福建医科大学分子生物中心完成;②SD大鼠88只,随机分成11组(n=8):假手术组、脊髓损伤组(简称:损伤组,包括伤后1天组、3天组、7天组、14天组、21天组)和损伤后移植干细胞组(简称:移植组,包括伤后1天组、3天组、7天组、14天组、21天组)。③改良Allen法建立大鼠SCI模型;④利用立体定位技术在脊髓损伤部位移植体外BrdU标记的NSCs;⑤损伤后用Gale联合评分法(the combine behavioral score,CBS)检测脊髓损伤大鼠行为改变;⑥免疫组化法检测SCI后TUNEL染色、bax及bcl-2的表达;⑦免疫荧光组织化学方法,观察NSCs的存活、整合等情况。 结果:①从新生大鼠脊髓分离的细胞群具有连续克隆能力,表达Nestin蛋白;②SCI后从14天开始,移植组CBS评分高于损伤组(p<0.05);③SCI后1天TUNEL染色明显,7天达高峰,14天仍较明显,21天降低;移植组凋亡指数(Apoptotic Index,AI)显著低于损伤组(p<0.05);④SCI后bcl-2、bax蛋白的表达均稳步上升(灰度值降低),14天开始下降;损伤组bcl-2与bax的灰度值之比(bcl-2/bax)大于移植组,即凋亡程度高于移植组(p<0.05)。⑤移植组和损伤组的bcl-2/bax与细胞凋亡指数AI做相关性分析两者均里显著性正相关;⑥BrdU标记的NSCs移植入SCI大鼠脊髓后21天,能存活。 结论:①改良Allen法所造成大鼠脊髓损伤属脊髓不完全损伤,是研究急、慢性脊髓损伤的较好模型;②神经干细胞在损伤脊髓内能存活,并与脊髓组织整合;③神经干细胞移植能提高脊髓损伤大鼠后肢功能恢复的程度;④神经干细胞可能通过促进bcl-2的表达、抑制bax的表达从而减少脊髓损伤后细胞凋亡,起保护作用。更多还原

【Abstract】 Objectives: To evaluate the functional recovery of acute spinal cord injuried rats treated with transplantation neural stem cells and to study the effects of apoptosis and expression of bcl-2、 bax on the experimental acute spinal cord injury and the neural stem cells that transplantated to it.Methods : In The center of molecule biology of Fujian Medical University, Neural stem cells were isolated, cultured and detected. Spinal cord injury (SCI) model was built by revised Allen method. The transplantation of BrdU-labeled cultured cells was performed immediately after injury. CBS scores were checked every day to show the motor function recovery. 1、 3、 7、 14、 21days postinjured the injured spinal cord was removed and detected by morphological observation and submitted to immunohistochemistry of the effects of apoptosis and expression of bcl-2、 bax on the experimental acute spinal cord injury and the neural stem cells that transplantated to it.Results: ①. Cells isolated from spinal cord of neonatal rats express Nestin antigen. ②. Animals treated with NSCs had pronounced higher CBS of hindlimb movements compared to controls after 14 days (p<0.05) .③. The expression of apoptosis positive cell showed increased tendency from 1st to 7th, and decreased at 21th. Animals treated with NSCs had pronounced lower lever of apoptosis index (AI) compared to controls (p <0.05). ④. The expression of bcl-2 and bax positive cell showed increased tendency from 1st to 7th, and decreased at 14th. The Apoptosis level is lower in transplantation gloup than that in no-transplantation gloup. ⑤bcl-2/bax positively correlate to AI inthe two groups. (6). large numbers of BrdU-labeled stem cells were observed live in spinal cord after 21days.Conclusions:?. Spinal cord injury (SCI) model built by revised Allen method is a incomplete contusive spinal cord injury and is suit to use in study acute or chrinic SCI in rat . (2). NSCs can survive and engraft into injuried spinal cord in rats. ?. NSCs can improve pronounced higher CBS score of hindlimb movements compared to controls. ?NSCs play an important role in the incomplete contusive spinal cord injury by inhibiting apoptosis that NSCs can improve the expression of bcl-2 and inhibit the expression of bax .更多还原