【作者】 张戈；

【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：观察联合应用ROCKII和GSK-3β抑制剂对成体大鼠脊髓损伤后轴突再生与功能恢复的影响。方法：(1)健康雌性SD成年大鼠185只,其中175只大鼠采用WD法制作成T1o平面完全性截瘫的脊髓损伤动物模型,并在L4平面蛛网膜下腔植入注药导管后随机分为5组,每组35只。A组为TDZD-8注射组,B组为Y27632注射组,C组为TDZD-8+ Y27632联合注射组, D组为PBS对照组,E组为手术对照组；F组10只大鼠,仅咬开椎板,不损伤脊髓,作为空白手术对照组。所用试剂均在手术后1h内经导管鞘内注射。(2)脊髓损伤后各组：在8h、24h、1w三个时间点,采用TUNEL法检测脊髓细胞凋亡；在24h、1w、2w三个时间点,检测生长相关蛋白-43(Growth-associated protein-43, GAP-43)的表达；在3w、6w、8w三个时间点,用BBB评分评估各组大鼠后肢运动功能；在3w、8w两个时间点,测定各组大鼠SEP；在8w采用顺行示踪的方法评估损伤节段脊髓轴突再生。结果：在各观察时间点：(1)A组至E组平均凋亡细胞数均高于F组,A组至C组在各观察时间点均低于D、E两组,C组在各观察时间点均低于A、B两组,差异均有统计学意义。(2)A至E组GAP-43的表达均高于F组,A组至C组GAP-43的表达在各观察时间点均高于D、E两组,C组GAP-43的表达在各观察时间点均高于A、B两组,差异有统计学意义。(3)顺行示踪,F组荧光均匀、排列有序,A组至E组荧光分布不均,A、B、C三组平均荧光面积高于D、E两组,其中C组最高,差异均有统计学意义；A组与B组之间, D组与E组之间,差异无统计学意义。(4)A组至E组BBB评分在各观察时间点均低于F组,A、B、C组评分高于D、E组,差异有统计学意义；A、B、C三组之间的差异无统计学意义,此结果可能是由于L4平面植入的导管造成的持续性压迫损所致。(5)A组至E组SEP潜伏期在各观察时间点长于F组,振幅低于F组,差异均有统计学意义；A、B、C三组之间及D、E两组之间潜伏期差异无统计学意义,并且它们之间振幅的差异也无统计学意义,此结果也可能是由于L4平面植入的导管造成的持续性压迫损所致。结论：(1)Y27632和TDZD-8均可减少脊髓损伤后细胞凋亡、使GAP-43表达增强,促进脊髓损伤后轴突再生与功能恢复。(2)联合应用Y27632和TDZD-8比单独应用的作用更明显。更多还原

【Abstract】 Objective:To explore the effect of neurite outgrowth and function recovery with co-application of ROCKII and GSK-3βinhibitors after spinal cord injury in rats. Methods:(1) 175 adult female Sprague-Dawley (SD) rats were randomly allocated into 5 groups after subjected to weight-drop impact causing complete paraplegia at T10 level and cathetered into subarachhnoid space via L4 level in each rats.Group A was treated with TDZD-8, Group B with Y27632, Group C with co-application of TDZD-8 and Y27632, Group D with PBS and Group E was sham operation group.The other 10 normal rats in group F was normal control group.Agent was injected into subarachhnoid space after SCI within 1 hour respectively (TDZD-8 with lmg/kg.d for 3 weeks,Y27643 with 1600ug/d for 2 weeks, TDZD-8 with lmg/kg.d for 3 weeks plus Y27643 with 1600ug/d for 2 weeks and PBS with 30uml/d for 3 weeks). In each group:the apoptosis of injured spinal cord was measured by TUNEL assay at 8,24hours and 1 week respectively and the expression of GAP-43 around the injured spinal cord was measured at 24hours,lweek and 2 weeks respectively.The BBB scors for hind limb motor function was evaluated at 3,6 and 8 weeks respectively and The SEP was measured at 3 and 8 weeks respectively.The neurite outgrowth was evaluated by anterograde tracing at 8 weeks. Results:The average cell numbers of apoptosis at 8,24hours and 1week in group F was fewer compared with group A to group E and it in group A to group C was fewer than in group D and group E respectively,and group c was the fewest of the five groups.There were no statistical different between group A and group B,and also between group D and group E.(2)The average positive areas of gap-43 expression at 24h,lw and 2w after SCI of group F was fewer than other groups. And group A to group C were wider than group D and group E,and group C was the widest of the five groups. Between group A and B,group D and E there were no statistical different.(3)From the normal group(group F),filaceous fluorescence with orderly distribution could been seen.Filaceous fluorescence or punctiform fluorescence with unorderly distribution could been seen in other groups. The average fluorimetric area of group A to group C was wider than group D and group E,and group C was the widest of the five groups.Between group A and B,group D and E there were no statistical different.(4)The score of normal rats was 21.The BBB scores of group F was significantly higher than other five groups.And group A,group B,or group C were higher than group D or group E.There were no statistical different among the group A,group B and group C,and between group D or group E. It might be due to the continuing compression caused by implanting the catheter into subbarachhonoid via L4 level.(5)Form group A to group E,the latent period was longer than the normal group(group F),and the amplitude was lower.The difference among the five groups(group A to group E)was similar to the BBB scores. Conclusion: co-application of Y27632 and TDZD-8 can are more effective of inhibiting apoptosis,alleviate secondary spinal cord injury, enhancing the expression of gap-43,promoting neurite outgrowthand facilitating the function recovery after SCI compared with Y27632 and TDZD-8 solely does.更多还原