【作者】 潘文明；

【导师】 唐天驷；

【作者基本信息】 苏州大学 ， 骨外科， 2010， 博士

【摘要】 第一部分大鼠急性脊髓损伤动物模型的建立与评价目的:建立大鼠脊髓损伤钳夹模型,观察假手术组,单纯脊髓损伤组,3-MA注射组在脊髓损伤后病理组织学变化和区别。方法:构建大鼠胸椎T8钳夹模型,用BBB评分法评价脊髓损伤后后肢运动功能变化,HE染色法和尼氏染色法观察各组脊髓损伤后的组织学变化。结果:大鼠胸椎T8钳夹损伤后,损伤脊髓出现明显的出血,水肿,髓鞘变性,空泡等,单纯脊髓损伤组与3-MA组比较在术后死亡率及后肢运动功能BBB评分上无统计学差异(p>0.05)。两组术后组织学评分在脊髓损伤程度上有统计学差异(p<0.05)。结论:1、成功建立大鼠脊髓钳夹损伤模型2、3-MA组在脊髓损伤后的术后3D时病理变化程度大于正常损伤组。第二部分细胞自噬及其相关基因Beclin-1,MAP1-LC3B在大鼠脊髓损伤组织中的表达目的:观察大鼠脊髓损伤后损伤区细胞自噬是否存在,比较不同时间点细胞自噬的表达情况。方法:将大鼠随机分为两组,每组各五个时间点:8H,1D,3D,7D,21D。损伤组按照第一部分的方法制作脊髓损伤模型,对照组只打开椎板而不损伤脊髓,按照上述时间点处死后,用透射电镜观察细胞自噬,用RT-PCR检测自噬相关基因Beclin1,LC3B的mRNA表达情况,用免疫组织化学和Western blot法检测自噬相关蛋白Beclin1,LC3B的表达情况。结果:透射电镜发现脊髓损伤后的各个时间点,在损伤的脊髓组织的神经元细胞?和胶质细胞中均可以发现自噬体,脊髓损伤后8H开始,细胞自噬相关基因Beclin1,LC3B的mRNA表达即较对照组明显升高,并且随着时间持续升高,在伤后3D时达到高峰,一直到伤后21D均有表达,而自噬相关蛋白Beclin1,LC3B的表达和其mRNA表达同步。结论:脊髓损伤后损伤区的神经元细胞和胶质细胞均有自噬性细胞死亡的表达,自噬相关基因Beclin1和LC3B从损伤后8H开始即表达量升高,到3D时到达高峰,21D时仍然存在。自噬相关蛋白Beclin1和LC3B表达与基因表达同步,较对照组明显升高。第三部分自噬抑制剂3-MA对大鼠脊髓损伤后神经细胞自噬及凋亡的影响目的:研究自噬抑制剂3-MA对大鼠脊髓损伤后自噬相关蛋白Beclin1和LC3B表达的影响,同时评价3-MA对大鼠脊髓损伤后细胞凋亡的影响。方法:将大鼠随机分成三组:对照组,单纯脊髓损伤组,3-MA干预组,每组同样分为伤后8H、1D、3D、7D、21D 5个时间点,使用荧光定量RT-PCR方法检测各组各个时间点Beclin1和LC3B基因表达的变化,使用荧光免疫组织化学方法评价各组各时间点Beclin1和LC3B蛋白表达的变化,使用TUNEL法和脊髓组织的DNA-Ladder比较各组脊髓损伤后细胞凋亡的程度,使用Westren-blot法检测各组凋亡相关蛋白Bcl-2和Bax的表达变化。结果:在我们观测的的五个时间点上,3-MA组较单纯脊髓损伤组Beclin1和LC3B的mRNA表达有明显降低,差异有统计学意义(P<0.05)。3-MA组的Beclin1和LC3B的蛋白表达也较单纯损伤组明显降低,差异有统计学意义(P<0.05)。脊髓损伤后可检测到明显的细胞凋亡,3-MA组的TUNEL阳性细胞数明显大于单纯损伤组,组间差异有统计学意义(P<0.05)。3-MA组脊髓组织的DNA-Ladder也较单纯损伤组重。凋亡相关蛋白Bcl-2的表达量为3-MA组最低,单纯脊髓损伤组其次,而对照组最高,组间差异有统计学意义(P<0.05)。凋亡相关蛋白Bax的表达量为3-MA组最高,单纯脊髓损伤组次之,对照组最低,组间差异有统计学意义(P<0.05)。结论:细胞自噬抑制剂3-MA可以明显抑制大鼠脊髓损伤后自噬相关蛋白及基因Beclin1和LC3B的表达。同时3-MA可以通过抑制自噬加重脊髓损伤后细胞凋亡的程度,因此脊髓损伤后细胞自噬是一种细胞的自我保护机制,可以拮抗脊髓损伤后的细胞凋亡。更多还原

【Abstract】 PartⅠEstablishment And Evaluation of Acute Spinal Cord Injury Model in Adult RatsObjective: To build an animal model of spinal cord injury by clamp force in adult rats, then to observe the changes and differences in locomotor scores and histopathological after spinal cord injury(SCI) in the three groups: Group Sham.Group SCI and Group 3-MA.Methods: Build a spinal cord injury model in dorsal transsection model (T8) by clamp force,The Basso-Beattie-Bresnahan (BBB) scores system is used to evaluate change of the locomotor scores after SCI, meantime, the HE(Hematoxylin and Eosin) staining and the Nissl staining are used to observer the histopathological change after the SCI.Results: There was significantly haemorrhage, edema, degeneration, vacuole and so on after spinal cord injury in rats with T8 dorsal transaction .The BBB scores of rats have no significant difference between Group SCI and Group 3-MA after SCI(p>0.05).The histopathological scores has significant difference between Group SCI and Group 3-MA after SCI (p<0.05)Conclusion: 1、Establish a spinal cord injury model in adult rats successfully .2.The histopathological changes of Group 3-MA is more than the Group SCI at the time point of 3 days after the operation。 PartⅡThe Expression of autophagic genes: Beclin-1 and MAP1-LC3B in lesion tissues after spinal cord injury in ratsObjective: TO investigate whether autophagy occurred in the spinal cord tissue after spinal cord injury(SCI).We also aimed to compare the expression value of autophagic correlative gene and Protein: Beclin1 & LC3B in different time point after SCI.Methods: Seperated the rats into two groups randomly :SCI group and control group. Each group had five time point. We built the SCI model using the way mentioned in the PartⅠ,while the rats in control group experience the same operation without injurying the spinal cord. At every time point, we used the transmission Electron Microscope to observe the ultrastructure of spinal cord tissue in order to find the autophagosome. The mRNA expression of Beclin1 and LC3B were also detected by the RT-PCR technique. The Immune-Histochemical(IHC) and Western-blot were used to measure the protein expression of Beclin1 and LC3B in every time point after SCI.Result: We found that the autophagosome could be seen in both the Neuron cell and the Glial cell at every time point after SCI. The mRNA expression value of Beclin and LC3B were raised since 8H Post-SCI and peaked at 3D post-SCI, and they could be detected until 21D after SCI. The protein’s expression curve of Beclin1 and LC3B were the same as the gene’s .Conclusion: The autophagic cell death could be detected in spinal cord injury region, both the neuron cell and glial cell experienced autophagic cell death. Compare to the contro group, the autophagic correlative gene and protein:Beclin1 and LC3B were found up-regulation from 8H to 21D after SCI, while peaked at 3D post-SCI. PartⅢ:The influence of 3-MA in autophagy and apoptosis after Spianl cord injury in rats.Objective:Aim to evaluate how the 3-MA ,which is a accredited autophagic inhibitor, effected the genic and proteinic expression of Beclin1 and LC3B. Meanwhile we wished to investigate the effect of cell apoptosis after spinal cord injury by the 3-MA.Methods: We separated the rats into three groups randomly, there were Group control, Group SCI, Group 3-MA. Each group had five time period, they are 8H.1D.3D.7D.21D after SCI. In this time point, We used the SYBR GREEN RT-PCR technique to measure the mRNA’s differences of Beclin1 and LC3B within the three groups. The proteinic differences of Beclin1 and LC3B within this three groups were evaluated by the Fluorescence immunohistochemistry. The level of apoptosis after SCI in the three groups were measured by the TUNEL. DNA ladder. The expression of Bcl-2 and Bax, which are closely interrelated with apoptosis, were tested by the Western-blot.Result: In the five time periods we observed, the genic expression of Beclin1 and LC3B were lower in the group 3-MA than in the group SCI, of course they are also higher than in the group Control. Also the differences between each two groups had significant difference(p<0.05). The proteinic expression of Beclin1 and LC3B in Group 3-MA were also lower than it in Group SCI, and they had significant difference(p<0.05). We found the apoptosis after SCI, and the TUNEL positive cell were much more in group 3-MA than it in Group SCI, the differences had statistical significance(p<0.05).The degree of DNA-Ladder were higher in Group 3-MA than in Group SCI. The proteinic expression of Bcl-2 is highest in group control, higher in group SCI and lowest in the group 3-MA. The difference in groups had significant difference(p<0.05). The proteinic expression of Bax is highest in group 3-MA, higher in SCI and lowest in group control. The difference in groups had significant difference(p<0.05).Conclusion: 3-MA as a accredited autophagic inhibitor, could both inhibited the ?genic and proteinic expression of Beclin1 and LC3B. Meanwhile ,the 3-MA could aggravate the apoptosis after SCI in rats by the way of inhibiting autophagy. So the autophagy could recognized as a mechanism of cellular self-protect, and autophagy played a antagonistic effect against apoptosis.更多还原