【作者】 张平；

【导师】 李振中；

【作者基本信息】 山东大学 ， 神经生物学， 2012， 硕士

【摘要】 目的：神经病理性疼痛(neuropathic pain, NP)是由于躯体感觉系统的损伤或疾病所引起的疼痛。NP作为慢性疼痛的一种,由于发病率高,严重影响患者生活质量,目前已受到越来越多的关注和重视。然而,NP的发病机制复杂,其治疗效果受到限制。促生长激素神经肽(galanin, Gal)与多种生物功能有关,如疼痛、发育和营养等。Gal可通过作用于Gal1型受体(galanin receptor1,GalR1)和Gal2型受体(galanin receptor2, GalR2)而发挥生物学效应。大量研究表明在神经损伤状态下,背根神经节(dorsal root ganglion, DRG)和脊髓灰质后角(spinal dorsal horn, SDH)内的Gal表达量上调,发挥抗疼痛、神经营养和促进神经再生等作用。本实验利用大鼠坐骨神经夹伤模型来研究外源性Gal对坐骨神经夹伤大鼠DRG和SDH神经元内Gal及其受体表达的调节,在行为学、坐骨神经功能学和形态学等方面的变化探讨外源性Gal对神经损伤的保护作用及机制。通过本实验为NP的实验研究、神经损伤的临床治疗提供一定理论基础和实验依据。方法：将63只鞘内置管成功的大鼠随机分成3组,每组21只：(1)夹伤组,即大鼠坐骨神经夹伤后每天鞘内注射10μ1人工脑脊液(artificial cerebrospinal fluid,ACSF)；(2)夹伤+Gal组,即大鼠坐骨神经夹伤后每天鞘内注射3μg Gal；(3)对照组,即大鼠假手术后每天鞘内注射10μl ACSF。术后每天进行行为学检测,并分别在术后8d、16d和24d,进行神经传导速度(nerve conduction velocity, NCV)测定,之后处死取材,再分别检测各组Gal及其受体蛋白及mRNA表达水平,利用甲苯胺蓝染色和透射电镜观察坐骨神经形态变化情况。结果：(1)通过行为学检测发现,坐骨神经夹伤可引起大鼠触觉异常性疼痛和热痛阈值降低,而鞘内注射外源性Gal可显著升高该阈值,并且可以使其提前恢复至正常水平。(2)Western blot和Real-time RT-PCR结果显示,与对照组相比,夹伤组大鼠DRG和SDH内Gal均明显升高；与夹伤组相比,夹伤+Gal组大鼠DRG内Gal表达明显下降,而SDH内Gal表达未见明显变化。夹伤组大鼠DRG内GalR1表达明显低于对照组,而SDH内GalR1明显上调；鞘内注射Gal使坐骨神经夹伤大鼠DRG和SDH内GalR1继续降低。与对照组相比,夹伤组大鼠DRG和SDH内GalR2均明显升高；鞘内注射Gal后,其表达未见明显变化。(3)利用肌电图检测、甲苯胺兰染色和透射电镜观察受损部位坐骨神经传导速度和形态结构变化发现,外源性Gal可显著改善受损坐骨神经的修复和再生。结论：鞘内注射外源性Gal减轻大鼠坐骨神经夹伤引起的触觉异常性疼痛和热痛觉过敏,并且促进损伤坐骨神经的修复,因此外源性Gal可以减轻神经损伤引起的NP并且具有明确的神经保护作用。在正常状态、坐骨神经夹伤和坐骨神经夹伤鞘内注射Gal等不同状态下,Gal及其受体mRNA和蛋白的表达有较强的可塑性,表明Gal及其受体参与了大鼠神经病变的病理生理过程。本研究的结果表明,对Gal及其受体系统进行干预可能对NP具有潜在治疗作用。更多还原

【Abstract】 Purpose:Neuropathic pain (NP) is a consequence of injury or disease affecting the somatosensory system. NP threats the health of human seriously, however its mechanism is still unknown completely and there is no effective treatment. It is well known that there will be great changes of neuropeptides after injury in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH), which causes NP. Neurotrophic peptide galanin (Gal) is strongly linked to several biological functions such as pain, development and trophic effects. A great deal of experimental data shows that Gal in DRG and SDH is upregulated in the state of nerve injury and play functions in antinociception, neurotrophy and neural regeneration. In this experiment, sciatic nerve-pinch injury rat model is used to study the functions of Gal and its receptors (GalR1, GalR2) in the state of nerve injury. It is wished to supply accurate and reliable theoretical basis for researches in neuropathic pain and clinical treatment for nerve injury. Materials and methods:Sixty-three rats catheterized successfully were randomly divided into3groups (21rats in each group). The first group is that rats with sciatic nerve-pinch injury were treated with vehicle solution. The second group is that rats with sciatic nerve-pinch injury were treated with Gal. The third group is that sham-operated control rats were just treated with vehicle solution. Then the threshold responses to mechanical and thermal stimuli were recorded everyday. Seven rats of each group were killed for tissue collection after NCV measurement at the day of8,16, and24(each time point7rats) after sciatic nerve-pinch injury. Real time-PCR and Western blot assay were used to analyze the levels of Gal, GalR1, and GalR2in L4-5DRG and the corresponding SDH. What is more, morphology of sciatic nerve was observed by toluidine blue staining and electron microscopy. Then the data were processed for statistical analysis. Results:(1) Exogenous Gal could release the NP in rats with sciatic nerve-pinch injury.(2) Gal increased significantly in DRG and SDH after sciatic nerve pinch injury. Gal administration downregulated Gal in DRG, but not SDH, after sciatic nerve pinch injury. GalR1decreased significantly in DRG after sciatic nerve pinch injury. Gal administration downregulated GalRl in DRG after sciatic nerve pinch injury as compared with that in sciatic nerve-pinched animals without Gal treatment. GalR1increased significantly in SDH after sciatic nerve pinch injury. Gal administration decreased GalR1in SDH after sciatic nerve pinch injury. GalR2increased significantly in DRG and SDH after sciatic nerve pinch injury. Gal administration did not have significant effects on GalR2expression in DRG and SDH after sciatic nerve pinch injury.(3) Exogenous Gal could improve sciatic NCV and nerve regeneration in rats with sciatic nerve-pinch injury. Conclusion:Gal and its receptor system take part in the pathophysiological processes of NP and nerve regeneration. In this model, by activating its receptors Gal release NP and promote nerve regeneration, which plays a protective role in nerve injury. These findings remind that it may be a potential way for curing NP to interfere Gal and its receptor system.更多还原