电针对大鼠脊髓损伤后细胞凋亡相关因子及神经再生的影响

【作者】 时素华；

【导师】 李志刚；

【作者基本信息】 北京中医药大学 ， 针灸推拿学， 2011， 博士

【摘要】 文献综述和实验研究两部分组成了本论文。第一部分文献综述第一篇重点介绍了近年来国内外脊髓损伤的研究进展,主要从脊髓损伤的机制和治疗(西医、中医、其他疗法)等方面来进行评述；第二篇论述了脊髓损伤后细胞凋亡、髓鞘变化、线粒体与脊髓损伤的关系；第三篇总结了应用电针干预脊髓损伤(SCI)的实验概况和临床研究概况。第二部分动物实验目的：探讨电针干预脊髓损伤(SCI)的治疗保护作用及其神经再生的机理。方法：随机将大鼠分为模型组、空白组、西药组、电针组,脊髓损伤的动物模型,我们采用改良式的Allen’s打击法来制作,分别于脊髓损伤后6h、1d、7d进行取材。(1)大鼠脊髓损伤后的运动、感觉等神经功能,我们采用运动联合计分法(CBS)来综合评定；(2)取模型组、空白组、西药组、电针组的损伤的局部脊髓组织固定、包埋、切片、染色后进行图像分析,测定损伤后局部脊髓组织内Fas、TNFR1的表达；(3)取模型组、空白组、西药组、电针组的损伤局部脊髓组织,提取总RNA,经RT-PCR反应、电泳成像与数据分析等过程,测定损伤后局部脊髓组织内Fas、TNFR1、caspase-3、calpastatin mRNA的表达。(4)透射电子显微镜下观察损伤局部脊髓组织髓鞘、线粒体的超微结构变化。结果：脊髓损伤后6h、1d、7d,(1)与空白组比较,模型组的CBS分值有极显著性差异(P<0.01)；与模型组比较,西药组和电针组的CBS分值,经统计学分析,有极显著性差异(P<0.01)。(2)电针组和西药组均能显著降低脊髓损伤6h、1d、7d后脊髓组织中Fas、TNFR1的表达。与模型组相比较,西药组和电针组Fas、TNFR1表达均降低,经统计学分析,并都有显著性差异(P<0.05)；西药组与电针组相比较,电针组Fas、TNFR1表达略高于西药组,经统计学分析,但无显著性差异(P>0.05)。(3)电针组和西药组均能显著增加脊髓损伤6h、1d、7d后脊髓组织中Calpastatin mRNA的表达。与模型组相比较,西药组和电针组Calpastatin mRNA的表达表达均升高,经统计学分析,并都有显著性差异(P<0.05)；西药组与电针组相比较,经统计学分析,无显著性差异(P>0.05)。(4)电针组和西药组均能显著降低脊髓损伤6h、1d、7d后脊髓组织中Fas、TNFR1、caspase-3 mRNA的表达。与模型组相比较,西药组和电针组Fas、TNFR1、caspase-3 mRNA表达均降低,经统计学分析,并都有显著性差异(P<0.05)；西药组与电针组相比较,经统计学分析,无显著性差异(P>0.05)。(5)透射电子显微镜显示：与模型组比较,电针组和西药组的脊髓组织,髓鞘更致密,变性现象减少,轴索内可见线粒体,但髓鞘板层分离,部分轴突水肿、凝固变性。结论：电针(督脉)对脊髓损伤的治疗作用可能是明显降低不同时段的Fas、TNFR1的表达,从而抑制脊髓损伤的细胞凋亡、减少成纤维细胞的大量堆积-瘢痕形成,达到对神经细胞保护的重要作用。电针(督脉)对脊髓损伤的治疗作用还可能通过明显降低Caspase-3、升高Calpastatin表达,阻断凋亡信号的传导,抑制Calpain的活性,减少髓鞘脱失,保存线粒体的功能,促进脊髓组织损伤的恢复,达到电针(督脉)对脊髓损伤组织的保护作用。更多还原

【Abstract】 literature review and experimental research.First Section:literature ReviewThe paper have three literature summarys. The first article reviews the research progress of the spinal cord injury (SCI) from western treatment and treatment of traditional Chinese medicine (TCM) in the resent years at home and abroad. The second article introduces the relationships among the apoptosis myelin sheath changes、mitochondria and spinal cord injury; The third article summarizes the clinical research and experimental surveys on the spinal cord injury (SCI) in rats with the acupuncture and moxibustion.Second Section:Experimental ResearchObjective:To explore mechanism of the therapeutic and protective functions with electroacupuncture (EA) to the spinal cord injury (SCI) in rats.Methods:We divided the rats into four groups randomly:the Model Group, the Blank Group, the Drug Group, the EA group. We use the modified Allen’s methods to make into a spinal cord contusion of the model rats. (1) comprehensive evaluation on movement、sensation、reflexion、the neural functions of SCI by using the CBS. (2) To detect the TNFR1、Fas of the local i njuried tissues by photo analysis which were fixed, embed, sliced, processed, dyed. (3) The local injured tissues of the Model Group, the Blank Group, the EA Group and the Drug Group were removed to detect the contents of Fas、TNFR1、Calpastatin、Caspase-3 mRNA through RT-PCR. (4) To observe the ultrastructural changes of axons and mitochondria in spinal cord tissues after SCI with transmission electron microscopy(TEM)Results:after 6h、1d、7d of SCI, (1) Compared with the Model group, the scores of CBS in the EA Group and the Drug group were significant differently (P<0.01); Compared with the Blank Group, the scores of CBS in the Model Group was significant differently (P<0.01); (2) The contents of Fas, TNFR1 in the local injuried tissues were decreased significantly in the EA Group and the Drug Group on the 6h、Id and the 7d after SCI. Compared with the Model Group, there were statistical significance in the EA Group and the Drug Group (P<0.01). Compared with the Drug Group, the Fas、TNFR1 contents of local injuried tissues were increased slightly in the EA Group and there was no statistical significance between the Drug Group and the EA Group (P>0.05). (3) The Calpastatin mRNA content of local injuried tissues were increased significantly in the EA Group and the Drug Group on the 6h、1 d and 7d after SCI. Compared with the Model Group, statistical significance were found in the EA Group and the Drug Group (P<0.01). There was no statistical significance between the Drug Group and the EA Group (P>0.05). (4) The contents of Fas、TNFR1、Caspase-3 mRNA in local injuried tissues were decreased significantly in the EA Group and the Drug Group on the 6h、1 d and 7 d after spinal cord injury. Compared with the Model Group, statistical significance were found in the EA Group and the Drug Group (P<0.01). There was no statistical significance between the Drug Group and the EA Group (P>0.05). (5) Compared with the Model Group, the myelin sheath were more compact, degeneration were reduced, mitochon -dria were seen in axons, but still exited separation of myelin board layer, edema, solidification and degeneration of part axons.Conclusion:EA has the therapeutic and protective effects on the SCI. It maybe works through reducing the contents of Fas、TNFR1 in different times to inhibit apoptosis、reduce the formation of the scares due to the fibroblasts substantial accumulation to protect the nerve cells after SCI;EA has the protective effects may also be increasing the contents of Calpastatin and decreasing the contents of Caspase-3 to inhibit apoptosis, reduce the inflammatory, restrain thr Calpain activity, reduce myelin depigmentation, save mitochondrial function on the SCI in rats.更多还原