【作者】 雷飞；

【导师】 冯大雄；

【作者基本信息】 泸州医学院 ， 外科学， 2010， 硕士

【摘要】 目的：观察GSK-3β抑制剂对大鼠脊髓损伤后轴突再生与功能恢复的影响。方法：①取健康雌性SD成年大鼠115只,体重250±30g,分为四组。A组：TDZD-8治疗组(35只),B组：PBS治疗组(35只)C组：手术瘫痪对照组(35只),D组：空白手术对照组(10只)。WD法制成SD大鼠T9平面完全性截瘫的脊髓损伤动物模型,脊髓损伤后1h内经蛛网膜下腔注射TDZD-8(1mg/kg/Qd)和PBS(60μl),连续注射3w。②各组在损伤后8h、24h、7d(每个时相2只大鼠)用4%多聚甲醛灌注取材后作冰冻切片,行TUNEL法检测细胞凋亡情况。③各组在损伤后24h、7d、14d(每个时相2只大鼠)用4%多聚甲醛灌注取材后作冰冻切片,行SP法作免疫组化染色检测GAP-43的表达情况。④各组在损伤后3w,6w、8w(每个时相5只大鼠)作BBB评分,评估双下肢运动功能恢复情况。⑤各组随机抽取3只模型,在损伤后第6w经大脑感觉运动皮质区注射FITC荧光示踪剂,注入示踪剂2w后4%多聚甲醛灌注取材后作冰冻切片,观察FITC荧光示踪剂的传导情况。⑥各组随机抽取5只模型在损伤后3w、8w检测体感诱发电位(SEP)振幅与潜伏期的变化情况。结果：①凋亡细胞的表达：脊髓损伤后8h开始出现凋亡细胞。TDZD-8治疗组阳性细胞数量均明显低于PBS治疗组和手术瘫痪对照组(P<0.05),但高于空白手术对照组。术后8h各组阳性细胞数量依次为：50.27±4.74、58.93±2.46、58.47±2.09、8.27±1.44。术后24h各组阳性细胞数量依次为：45.73±4.95、55.40±3.94、54.53±3.11、8.27±1.44。术后7d各组阳性细胞数量依次为：10.67±1.50、15.67±1.49、15.60±1.68、8.27土1.44。②GAP-43的表达：脊髓损伤后GAP-43的表达逐渐增加,第14d达到高峰,各时间点中TDZD-8治疗组GAP-43表达面积均明显高于其余三组(P<0.05)。术后1d各组GAP-43阳性表达的面积依次为：79.2±5.31、43.3±3.99、42.3±4.77、6.3±2.31,术后7d各组GAP-43阳性表达的面积依次为：107.3±4.43、70.1±3.57、68.2±3.34、6.3±2.31,术后2w各组GAP-43阳性表达的面积依次为：156.7±4.35、94.5±3.91、96.1±3.56、6.3±2.31。③荧光示踪结果：损伤区域脊髓组织结构紊乱,TDZD-8治疗组有较多再生纤维穿过损伤部位,而PBS治疗组和手术瘫痪对照组损伤部位有少许的神经纤维再生,无法穿过损伤部位,再生神经纤维的面积计数依次为：82.0±1.7,35.2±1.7,33.74±1.3,138±2.2。④术后3w、8w检测SEP结果：TDZD-8治疗组较PBS治疗组、手术瘫痪对照组潜伏期明显缩短,振幅显著升高。⑤BBB运动功能评分：术后各组评分均有升高,术后8w PBS治疗组、TDZD-8治疗组、手术瘫痪对照组和空白手术对照组评分为：9.4±1.14、8.2±0.45、8.0±0.71、21±0.00。p<0.05结论：①.TDZD-8能够抑制神经细胞凋亡,减少脊髓继发性损伤。②.TDZD-8能够上调生长相关蛋白43,能够缩短体感诱发电位潜伏期、增加其波幅,促进残存神经元出芽或促进轴突再生,增加神经元的可塑性,促进脊髓损伤后功能恢复。更多还原

【Abstract】 Abstract:Objective:To explore the effect of promoted axonal regeneration and functional recovery with GSK-3βinhibitors in vivo after spinal cord injury in rats.Methods:①A total of 115 healthly female Sprague-Dawley (SD) rats (weight 250±30)were randomly allocated into TDZD-8 group、PBS group、SCI group and Sham operated group with 10 rats in Sham operated group and 35 in other groups。The laminectomy was performed and a weight-dropoing impact at spinal levels of T9 to cause complete paraplegia。TDZD-8 or PBS were injected at the dose of (1mg/kg/Qd) or (60μl/Qd) through subarachnoid one hour after SCI and continued for three weeks。②The rats were killed by perfusion with 4%paraformaldehyde at 8h、24h、7d after SCI(2 rats at each time phase),then the frozen sections were stained with TUNEL method to measure the degree of apopotic。③The rats were killed by perfusion with 4%paraformaldehyde at 24h、7d、14d after SCI(2 rats at each time phase),then the frozen sections were stained with SP immunohistochemical method to measuring the expression of GAP-43。④Rats were used to make BBB scale in order to evaluate lower limb function recovery at 3w、6w、8w after SCI(5 rats at each time phase)。⑤Six weeks after SCI, three rats were injected FITC fluorescent tracer from sensorimotor cortex in each group。Two weeks after FITC inject, the animals were killed by perfusion with 4% paraformaldehyde, then the frozen sections were observed by Confocal microscopy in order to know the FITC fluorescent tracer passing condition。⑥Three weeks and Eight weeks after SCI, five rats were choosen to detect amplitude and latency changes of somatosensory evoked potential in each group。Results:①At the eight hours after SCI,the apoptosis positive cells were observed。The positive cells of TDZD-8 group were significantly lower than PBS group and SCI group, but higher than the Sham operated group.Eight hours after SCI,the positive cells of each group are 50.27±4.74,58.93±2.46、58.47±2.09、8.27±1.44 in turns。Twenty-four hours after SCI,the positive cells of each group are 45.73±4.95、55.40±3.94、54.53±3.11、8.27±1.44 in turns。Seven days after SCI,the positive cells of each group are 10.67±1.50、15.67±1.49,15.60±1.68,8.27±1.44 in turns。②After SCI, the GAP-43 positive reaction was increased gradually,and rose to its peak on the 14th day, which in TDZD-8 group was remarkably superior to that in other groups in different time point (P<0.05)。One day after SCI,the positive area of each group are 79.2±5.31、43.3±3.99、42.3±4.77、6.3±2.31 in turns,Seven day after SCI,the positive area of each group are:107.3±4.43、70.1±3.57、68.2±3.34、6.3±2.31 in turns,Two weeks after SCI,the positive area of each group are:156.7±4.35、94.5±3.91、96.1±3.56、6.3±2.31 in turns。③The structure of regenetation axonals are significantly disordered in lesion areas,TDZD-8 group have more regenetation axonals extend into caudal while the PBS group and SCI group have little regenetation axonal extend into caudal。The areas of regenetation axonals was followed:82.0±1.7,35.2±1.7,33.74±1.3,138±2.2。④Somatosensory evoked potentials examination postopertative 3、8 week:Compared TDZD-8 group with PBS group and SCI group,The waveform of somatosensory evoked potentials was significantly short, and the amplitude was significantly high。⑤BBB locomotor scale:All the BBB scores have arised, The mean of BBB scores of four group at 8 weeks after SCI were 9.4±1.14、8.2±0.45、8.0±0.71、21±0.00。Among the groups,there was significant difference between TDZD-8 group with PBS group and SCI group(p<0.05)。Conclusion:①TDZD-8 can inhibit neuronal apoptosis after spinal cord injury and reduce secondary spinal cord injury。②TDZD-8 can increase the growth associated protein 43 expression, reduce latency of somatosensory evoked potentials and improve the amplitude,promote the survival neuron sprouting or promote axonal regenaration,increase the plasticity of neurons,promote functional recovery。更多还原