【作者】 杨林；

【导师】 吕德成；

【作者基本信息】 大连医科大学 ， 外科学， 2016， 博士

【摘要】 急性脊髓损伤(Acute Spinal Cord Injury,ASCI)是一类非常严重的神经系统疾病,常导致损伤平面之下的运动和感觉功能全部或部分丧失。脊髓损伤患者一生需要的治疗费用非常昂贵,治疗效果又不理想,所以给个人和家庭都带来了巨大的经济和心理负担。因此,怎样提高治疗急性脊髓损伤的效果,恢复或者减少其所造成的神经功能损害,让患者重新回归到正常的社会生活工作中来,具有非常重要的意义。他克莫司(Tacrolimus,FK506)商品名普乐可复,已经通过美国FDA验证,1984年由日本藤泽制药公司在筑波泥土的链霉菌里提取出来的发酵产物,其属于大环内酯类抗生素,临床上具有很强的免疫抑制作用。据文献报道,FK506的受体蛋白FKBP(FK506 Binding Protein)不但存在于免疫系统中,并且在神经系统内的也极为丰富,并是前者的10~50倍。但是,他克莫司作为免疫抑制类药物虽然具有神经营养和保护的作用,同时也具有神经毒性作用,研究表明其神经毒性作用与药物的浓度有关,所以如何在发挥神经营养和神经保护作用的同时避免产生神经毒性作用是一个难点。目前关于FK506的神经营养与神经保护的作用机制尚不清楚,同时也没有关于他克莫司对脊髓运动神经元缺血缺氧损伤保护作用方面的研究。实验中我们通过成功建立新生大鼠脊髓运动神经元的培养体系,进而筛选适宜运动神经元生长的最佳他克莫司药物浓度。在获得最佳药物浓度后构建脊髓运动神经元缺血缺氧模型,设立对照组和药物组,观察并研究他克莫司的神经营养及保护作用并阐述其相关的机制。本研究填补了目前没有关于他克莫司对于脊髓运动神经元缺血缺氧保护作用研究的空白,同时也为他克莫司的神经营养作用提供进一步的研究支持,为临床上利用他克莫司治疗急性脊髓损伤疾病给予了新的理论依据。第一部分新生大鼠脊髓运动神经元细胞的纯化培养与鉴定目的:探讨新生大鼠脊髓运动神经元的分离、纯化和培养方法,并予以分类鉴定,测定脊髓运动神经元纯度及存活率,建立新生大鼠脊髓运动神经元的纯化培养体系。方法:取新生大鼠的脊髓腹侧组织分离消化成细胞悬液,经密度梯度离心及差速贴壁后纯化培养,5d后运用免疫细胞化学双标染色法对培养盖片上的细胞进行分类和鉴定,计数随机50个视野中各细胞成分的平均含量。初接种后随机选取50个视野进行细胞计数,以0d的细胞数目均值作为基数100%。继续在培养后的1、3、5d各时间点用上法进行细胞计数,并进行存活率计算及统计学分析。结果:细胞的生长状况良好,神经元细胞占细胞总数的85.8%。其中,运动神经元细胞占总数的71.6%,星形胶质细胞占总数的7.8%,非神经元细胞占总数的6.4%。神经元细胞的存活数目较多,5d后神经元细胞的存活率仍保持在57.8%左右。结论:取新生大鼠的腹侧脊髓组织并结合密度梯度离心法和差速贴壁接种法可以培养出高纯度的脊髓运动神经元,同时脊髓运动神经元的高存活率也保证了应用运动神经元细胞进行相关实验的顺利进行。第二部分他克莫司对脊髓运动神经元保护作用及相关机制的实验研究目的:寻找他克莫司(FK506)促进脊髓运动神经元生长的最佳浓度,观察其对缺血缺氧的脊髓运动神经元有无保护作用,并探讨其可能的相关机制。方法:取已培养3d的脊髓运动神经元细胞分别加入不同浓度的他克莫司,分为:空白对照组(A组)、100pmol/L FK506组(B组)、1nmol/L FK506组(C组)和10nmol/L FK506组(D组),继续培养2d,通过细胞免疫化学法观察神经元细胞的生长情况,四甲基偶氮唑盐比色法(MTT法)检测神经元细胞的活力,逆转录聚合酶链式反应法(RT-PCR法)检测生长相关蛋白-43信使核糖核酸(GAP-43m RNA)的表达情况。在获得了FK506的最佳浓度后,另取一部分脊髓运动神经元分为对照组,缺血缺氧组,FK506治疗组,将已培养3d的缺血缺氧组和FK506治疗组的运动神经元细胞培养基换成无血清培养基,同时用石蜡油封闭各孔造成缺氧条件。缺血缺氧组在损伤的前一天每孔给予0.01mol/L p H7.2的PBS 800μl,FK506治疗组在损伤前一天每孔加入已得到的最佳浓度FK506 800μl,继续培养2d,对照组不做任何处理。通过免疫荧光法观察各组神经元细胞生长情况,MTT法检测各组神经元细胞活力,RT-PCR法检测各组神经元细胞GAP-43m RNA的表达,Western-blot法检测在加入FK506后各时间点ERK、p-ERK、GAP43等蛋白的表达变化。结果:1nmol/L FK506组(C组)的新生大鼠脊髓运动神经元数量和突起长度明显优于A组、B组和D组。同时C组的MTT吸光度OD值及GAP-43m RNA的相对表达量也明显高于A组、B组和D组,差异有显著统计学意义(P<0.01)。缺氧缺血造模后的免疫荧光结果显示,FK506治疗组的神经元数量和突起长度均明显优于缺血缺氧组,FK506治疗组的MTT吸光度OD值及GAP-43m RNA的表达量也均明显高于缺血缺氧组,具有显著统计学差异(P<0.01)。Western-blot结果显示,在加入FK506作用不同时间点后,FK506治疗组运动神经元p-ERK的表达量较缺血缺氧组均有显著增加,并在4小时后达到顶峰,而GAP-43的表达在8小时后达到顶峰。结论:对新生大鼠脊髓运动神经元生长促进和保护作用的FK506最佳浓度是1nmol/L。FK506可提高体外缺血缺氧损伤的新生大鼠脊髓运动神经元的细胞活性,并增加p-ERK和GAP-43在脊髓运动神经元中的表达。其中p-ERK开始表达的时间和达峰时间均早于的GAP-43,支持GAP-43为p-ERK下游表达分子的观点,提示缺氧缺血后ERK的活化可增加GAP-43的表达,而后者则具有促神经再生和神经修复的作用。第三部分他克莫司治疗大鼠急性脊髓损伤的实验研究目的:观察他克莫司(FK506)对大鼠脊髓损伤后神经功能的改善作用并探讨可能的相关机制。方法:随机将72只健康SD大鼠分成三组:假手术组、损伤模型组、FK506治疗组,其中损伤模型组和FK506治疗组采用Allen’s法击伤大鼠T10脊髓制造大鼠急性脊髓损伤模型,假手术组仅做椎板切除术。其中FK506治疗组于术后10分钟给予0.3mg/kg的FK506腹腔注射,之后每天注射1次,连续2周,其余两组均给予等体积的生理盐水。造模后每组根据取材时间进一步分为术后1、2、4、6h四小组,每小组6只,测定脊髓损伤处组织MDA、SOD、NO含量。造模后1、3、7、14、21d采用甲苯胺蓝染色观察脊髓组织病理学改变,并进行脊髓功能BBB评分及斜板实验评价大鼠神经功能恢复情况。结果:伤后1、3、7、14、21d FK506治疗组脊髓损伤区出血及坏死情况均较损伤模型组轻,而斜板实验和BBB评分均明显优于损伤模型组,两组间比较差异有统计学意义(P<0.05),损伤模型组与FK506治疗组MDA、SOD及NO含量在损伤后1h未见明显差异,而在损伤后2h、4h和6h显示FK506治疗组对急性脊髓损伤后MDA及NO含量的升高具有明显的抑制作用,对SOD含量的降低有明显的提升作用,具有显著统计学差异(P<0.01)。结论:在大鼠急性脊髓损伤后早期应用他克莫司可以减少神经细胞凋亡,减轻脊髓的继发性损伤,促进神经功能的恢复。其神经保护作用的发挥,可能与抑制损伤区炎症反应,提高脊髓细胞活力有关。更多还原

【Abstract】 Acute Spinal Cord Injury(Acute Spinal Cord Injury, ASCI) is a serious nervous system injury, often cause nerve damage below the injury level and part or all loss of sensory and motor functions. In the developed world, the patients with spinal cord injury costs average of $750000 or more for treatment, it bring great economic burden to individuals, families and society. Therefore, the treatment of acute spinal cord injury and make the patient back to normal social life has the vital significance.Tacrolimus(FK506), the commodity name is Prograf,has passed the FDA certification.It was isolated from the soil of fermentation product by Fujisawa pharmaceutical company, it belong to the macrolide antibiotics, have stro ng immunosuppressive effects. FK506 receptor protein-FKBP(FK506 Binding Protein) not only exists in the immune system,but also in the nervous system, it about 10 to 50 times of the former. The present study showed that tacrolimus as an immunosuppressive drug has neurotrophic and neuroprotective effects,but the drug’s neurotoxicity was related to the concentration, avoid to produce neurotoxic effect is difficult. At present the mechanism of nerve nutrition effects in FK506 is not clear, and there is no study talk about the protective effect of tacrolimus on hypoxia-ischemia spinal motor neurons. On the basis of training system of neonatal rats’ spinal motor neurons established, then we select the optimal concentration of tacrolimus to protect neurons’ growth. After optimal concentration of tacrolimus has been finded,we set up the hypoxia-ischemia’s model, observe the nerve nutrition and protection effects of tacrolimus and investigate its related mechanism.This research fills the blank of the research about tacrolimus affect for hypoxia-ischemia spinal motor neurons.At the same time, it provides support for the nutritional function of tacrolimus, which provides a new theoretical basis for clinical treatment of acute spinal cord injury.Part 1:Spinal motor neurons from neonatal rats: purification, culture and identificationObjective:To establish the culture system of spinal motor neurons from neonatal rats.Classification, identification and survival rates determination of spinal cord neuron.Method: Ventral spinal cord tissues from neonatal rats were made into cell suspension that was subjected to density gradient centrifugation and differential adherence followed by purification culture. Then, the cells on cover plates were identified and classified using immunocytochemical double staining method, and the content of cell components was calculated in combination with fluorescent Hoechst nuclear staining.Continue to develop 5 days, randomly selected 50 fields from each time point to calculate the number of living cells and did the statistical analysis.Results: The cells adhered the wall and grows well, the neurons accounted for 85.8%, motor neurons reached 71.6%, astrocytes accounted for 7.8%, cells negative for neurofilament 200 and glial fibrillary acidic protein were 6.4%.The number of the neurons were more,and the survival rate remained 57.8% after 5d.Conclusions:These findings indicate that the ventral spinal cord tissues from neonatal rats combined with density gradient centrifugation and differential adherence can develop high-purity spinal motor neurons. At the same time, the high survival rate of neurons, ensure the smooth progress of the follow-up experiments and successfully established a neonatal rats’ spinal motor neurons culture system.Part 2:Experimental study of the protective effect of tacrolimus on spinal motor neurons and its’ mechanismObjective:To find the optimal concentration of tacrolimus(FK506) for the growth of spinal cord motor neurons, observe the neuroprotective effect of the hypoxia-ischemia spinal motor neurons, and to explore the possible mechanism.Method: After the motor neuron cells were cultured for three days, we added different concentration of FK506 to classified the motor neurons into: blank control group(Group A), 100 pmol FK506 group(Group B), 1nmol/L FK506 group(Group C) and 10nmol/L FK506 group(Group D). Continue to culture two days, we use immunofluorescence staining to observe the growth of spinal motor neurons,MTT colorimetric assay to detect the vitality of spinal motor neurons,RT-PCR to detect the growth associated protein-43 m RNA expression of spinal motor neurons. After obtained the optimal concentration of FK506, we divided the spinal cord motor neurons into control group, hypoxia-ischemia group, FK506 treatment group.On the third day, the culture medium was aspirated and replaced serum-free medium in hypoxia-ischemia group and FK506 treatment group,close the hole with paraffin oil cause hypoxia condition. The control group without any treatment.In the hypoxia-ischemia group, we added 800μL 0.01 mol PBS per pore.In FK506 treatment group,we added 800μL optimal concentration of FK506 per pore,continue to culture two days.Then we use immunofluorescence staining to observe the growth of spinal motor neurons,MTT colorimetric assay to detect the vitality of spinal motor neurons,RT-PCR to detect the growth associated protein-43 m RNA expression of spinal motor neurons. Western blot to detect the motor neuron cell p-ERK protein expression after FK506 treatment.Results: The number and the length of the spinal cord motor neurons in group C were significantly better than group A, group B and group D. The OD value of MTT and m RNA relative expression level in group C was higher than group A, group B and group D, the difference has statistically significant(P<0.01). With optimal concentrations of FK506 treatment of spinal motor neurons immunofluorescence results showed that, the motor neuron number and neurite length in FK506 treatment group were significantly better than hypoxia-ischemia group, the OD value of MTT and GAP-43 m RNA relative expression levels in FK506 treatment group were significantly higher than hypoxia-ischemia group, has significant difference(P<0.01). According to the results of Western blot, the spinal motor neurons’ p-ERK expression were significantly increased, the peak time were 4 hours and the GAP-43 expression peak time were 8 hours.Conclusions:The optimal concentration of FK506 for neuroprotective effect is 1nmol/L. FK506 can improve the cell activity of hypoxia-ischemia spinal motor neurons in neonatal rats.Hypoxia-ischemia can increase p- ERK and GAP-43 protein expression in spinal motor neurons, the peak time of p-ERK protein expression were earlier than the GAP-43, support the view that GAP43 is downstream molecular of p-ERK,which have the function of nerve regeneration and repair.Part 3:Experimental study of tacrolimus in the treatment of acute spinal cord injury in ratsObjective:To observe the tacrolimus(FK506) in the improvement of nerve function after acute spinal cord injury in rats and explore the possible mechanism.Method:Randomly divided 72 healthy SD rats into three groups: control group,injury model group, FK506 treatment group.The injury model group and FK506 treatment group’s T10 spinal cord wounded by Allen’s blow device to make the acute spinal cord injury model, the control group only do laminectomy. 10 minutes after injury,the FK506 treatment group did the intraperitoneal injection with 0.3mg/kg FK506,once a day, during 2 weeks.The rest of the two groups were given equal volume of saline solution.According to the time after injury(1,2,4,6h) the rats in their group were divided into four teams, each team has 6 rat,measure the MDA, SOD and NO levels of spinal cord tissue. Using toluidine blue staining to observe spinal cord tissue pathology change,BBB score and inclined plate experiment to detect the spinal cord function at 1,3,7,14,21 days post- injury.Results: At 1, 3, 7,14,21 days post-injury,the situation of hemorrhage and necrosis in the spinal cord in FK506 treatment group were better than injury model group.The inclined plate test score and the BBB score in FK506 treatment group were higher than the injury model group, the difference between the two groups is significant(P<0.05).The content of MDA, SOD and NO in the injury model group and the FK506 treatment group had no significant difference at postoperative 1h, but at postoperative 2,4,6h,the FK506 can inhibited the contents of MDA and NO increase obviously, improved the content of SOD decreased significantly, has significant difference(P<0.01).Conclusions: Early application of tacrolimus can reduce the apoptosis of nerve cells, relieve secondary spinal cord injury, promote the recovery of spinal cord function. The protective effect may be related to inhibit the inflammatory reaction and enhance the cell viability of spinal cord.更多还原