去铁胺修复脊髓损伤的实验研究

发布时间：2018-06-16 14:41

本文选题：脊髓损伤 + 去铁胺　；参考：《天津医科大学》2017年硕士论文

【摘要】：【目的】脊髓损伤是骨科严重的创伤性疾病,目前无有效的治疗方式,给患者、家庭和社会带来严重负担。脊髓损伤后的炎症反应在继发性脊髓损伤过程中起到至关重要的作用,有研究报道去铁胺(Deferoxamine,DFO)对大鼠脊髓损伤具有一定的治疗作用,但是机制未完全阐明,本研究旨在探索DFO是否在大鼠脊髓打击损伤模型中对脊髓损伤后炎症反应起抑制作用而对脊髓损伤(Spinal Cord Injury,SCI)产生治疗作用。【方法】1.动物模型制作本研究采用Impactor Model-Ⅱ型脊髓损伤打击器(10 g×25 mm)制作大鼠脊髓胸10(T10)节段脊髓挫伤模型。2.动物实验分组实验动物采用8周龄雌性Wistar大鼠54只,实验动物随机均分为三组:Sham组,Injury组,Injury+DFO组。3.DFO治疗作用评价3.1.总铁离子含量检测于SCI后2 d和14 d 2个时间点对3个实验组脊髓使用总铁离子检测试剂盒检测局部总铁离子含量。3.2.炎症因子表达检测于SCI后2 d和14 d 2个时间点对3个实验组脊髓进行取材,Western Blot检测炎症因子肿瘤坏死因子(TNF-α)、白介素1-β(IL1-β)和凋亡蛋白Caspase-3的表达情况。3.3.脊髓病理结构评价于SCI后2 d和14 d 2个时间点对各实验组脊髓进行取材并制备脊髓石蜡切片,进行胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经元核心抗原(neuronal nuclei,NeuN)、2'3'环核苷酸磷酸二酯酶3(2'3'cyclic nucleotide3'phosphodiesterase,CNPase)免疫组织化学染色、苏木精-伊红染色(HE染色)和TUNEL法凋亡检测判断脊髓局部星形胶质细胞、神经元及少突胶质细胞、局部组织结构变化和局部细胞凋亡变化情况。3.4.大鼠后肢运动功能评价造模成功后,各实验组大鼠于给药后1 d、7 d、14 d、28 d、42 d、56 d使用BBB Scale对大鼠后肢运动功能恢复评分。【结果】1.成功建立大鼠脊髓挫伤模型造模后大鼠出现摆尾、后肢抽搐、脊髓打击部位出现红肿及出血。BBB评分显示在损伤后1 d所有实验组大鼠评分均为0分。2.总铁离子含量检测SCI后2 d,Sham组、Injury组和Injury+DFO组的总铁离子含量分别为8.930±2.124(μmol/gprot)、22.502±3.436(μmol/gprot)和9.141±1.062(μmol/gprot)。SCI 14 d后Sham组、Injury组和Injury+DFO组的总铁离子含量分别为8.785±2.671(μmol/gprot)、65.677±2.604(μmol/gprot)和13.009±1.272(μmol/gprot)。3.炎症因子表达检测SCI后2d,Sham组、Injury组和Injury+DFO组SCI局部IL1-β的相对表达水平分别为1±0.231、1.74±0.217和1.01±0.312;Sham组、Injury组和Injury+DFO组SCI局部TNF-α的相对表达水平分别为1±0.193、1.53±0.245和1.18±0.187;Sham组、Injury组和Injury+DFO组SCI局部Caspase-3相对表达水平分别为1±0.159、2.89±0.231和1.71±0.224。SCI 14d后,Sham组、Injury组和Injury+DFO组SCI局部IL1-β的相对表达水平分别为1±0.267、1.73±0.193和0.93±0.287;Sham组、Injury组和Injury+DFO组SCI局部TNFα的相对表达水平分别为1.02±0.291、1.39±0.177和0.97±0.169;Sham组、Injury组和Injury+DFO组SCI局部Caspase-3的相对表达水平分别为1±0.179、2.27±0.312和1.20±0.254。4.脊髓病理评价于SCI后2 d和14 d 2个时间点对各实验组脊髓进行取材并制备脊髓切片,进行胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、神经元核心抗原(Neuronal nuclei,Neu N)、2'3'环核苷酸磷酸二酯酶3(2'3'cyclic nucleotide 3'phosphodiesterase,CNPase)免疫组织化学染色、苏木精-伊红染色(HE染色)和TUNEL法凋亡染色,结果显示:相对于Injury组,Injury+DFO组明显的减少GFAP面积和TUNEL阳性细胞数量,并且增加CNPase阳性面积和NeuN阳性细胞数量,对SCI后脊髓病理结构有明显的修复作用。5.大鼠后肢运动功能评价Sham组在第0天的BBB评分为21分,而Injury和Injury+DFO组的评分均为0分,表明大鼠SCI模型的有效性。各组评分随时间逐渐升高,然而,在SCI后7天,Injury和Injury+DFO组之间的BBB评分具有统计学显着差异。在SCI后8周,Injury组的BBB评分为14,Injury+DFO组为18。结果表明,DFO对大鼠SCI后运动功能有明显的恢复作用。【结论】DFO可以显著减少SCI局部铁离子含量,减少SCI局部TNFα、IL1-β和Caspase-3等因子的表达,保护局部原有结构细胞,减少细胞凋亡,恢复大鼠后肢运动功能,促进SCI的修复,为SCI治疗提供新的药物治疗选择,为临床修复SCI提供实验依据。
[Abstract]:[Objective] spinal cord injury is a serious traumatic disease in the Department of orthopedics. There is no effective treatment for patients, family and society. The inflammatory reaction after spinal cord injury plays a vital role in secondary spinal cord injury. It is reported that Deferoxamine (DFO) has a certain effect on spinal cord injury in rats. The therapeutic effect, but the mechanism is not fully elucidated, the purpose of this study is to explore the effect of DFO on spinal cord injury (Spinal Cord Injury, SCI) in the spinal cord injury injury model in rats. [Methods] 1. animal models were made to use Impactor Model- II type spinal cord injury. The rat spinal cord thoracic 10 (T10) (T10) segment spinal contusion model was made by the attack device (10 G x 25 mm) in the animal experiment group of.2. animal experiment group and 54 female Wistar rats of 8 weeks of age were used. The experimental animals were randomly divided into three groups: Sham group, Injury group and Injury+DFO group.3.DFO therapeutic effect evaluation 3.1. total iron content of 3.1. was detected in 3 after SCI 2 D and 14 times 2 time points. In the experimental group, the total iron ion content of the spinal cord was detected by the total iron ion detection kit and the expression of.3.2. inflammatory factor was detected in the spinal cord of 3 experimental groups after SCI 2 D and 14 d. Western Blot was used to detect the inflammatory factor tumor necrosis factor (TNF- alpha), and the expression of interleukin 1- beta (IL1- beta) and apoptotic protein Caspase-3 was.3.3.. The spinal cord pathological structure was evaluated at the 2 time points of SCI 2 D and 14 d to prepare the spinal cord and prepare the spinal paraffin section for glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP), neuron core antigen (neuronal nuclei, NeuN), and 2'3'cyclic nucleotide phosphodiesterase 3. Erase, CNPase) immuno histochemical staining, hematoxylin eosin staining (HE staining) and TUNEL apoptosis detection to determine the local astrocytes, neurons and oligodendrocytes in the spinal cord, the changes of local tissue structure and the changes of local cell apoptosis in the.3.4. rat hind limb movement function evaluation, the rats in the experimental group were given the drug after administration. 1 D, 7 d, 14 d, 28 d, 42 d, 56 D used BBB Scale to evaluate the motor function recovery of the hind limbs. [results] 1. successfully established the rat spinal cord contusion model, the rats appeared the tail, the hind limbs twitching, the swelling of the spinal cord and the bleeding.BBB score showed that the scores of the rats in all the experimental groups of the 1 D were 0 fractions and.2. total iron ions after the injury. The total iron content in the 2 D, Sham, Injury and Injury+DFO groups were 8.930 + 2.124 (mu mol/gprot), 22.502 + 3.436 (mu mol/gprot) and 9.141 + 1.062 (mu mol/gprot).SCI 14 d, respectively. The total iron content of the Injury group and the group was 8.785 + 2.671 (MU), 65.677 + 2.604 (MU) and 13.009, respectively. The relative expression level of SCI local IL1- beta in 2D, Sham, Injury and Injury+DFO groups was 1 + 0.231,1.74 + 0.217 and 1.01 + 0.312, respectively, and the relative expression level of the Sham group and Injury+DFO group was 1 + 1 + 0.245 and 1.18 + 0.187 in the Sham group and the Injury+DFO group, respectively. The relative expression level of local Caspase-3 in group SCI and Injury+DFO group was 1 + 0.159,2.89 + 0.231 and 1.71 + 0.224.SCI 14d respectively. The relative expression level of SCI partial IL1- beta in Sham group, Injury group and Injury+DFO group was 1 + 0.193 and 0.93 + 0.287, respectively. The relative expression level of SCI local Caspase-3 in group Sham, Injury and Injury+DFO was 1 + 0.179,2.27 + 0.312 and 1.20 + 0.254.4. in the spinal cord pathology evaluation at 2 time points of 2 D and 14 d after SCI, and the spinal cord sections were prepared and prepared for the spinal cord, and the glial fibrillary acidic protein (glial) was carried out. Brillary acidic protein, GFAP), neuron core antigen (Neuronal nuclei, Neu N), 2'3'cyclic nucleotide phosphodiesterase 3 (2'3'cyclic nucleotide 3'phosphodiesterase), immunohistochemical staining, hematoxylin eosin staining and dying dying staining. The number of FAP area and TUNEL positive cells increased the positive area of CNPase and the number of NeuN positive cells. After SCI, the pathological structure of the spinal cord was obviously repaired in the.5. rat hind limb motor function. The BBB score of the Sham group in the Sham group was 21 at zeroth days, while the scores of the Injury and Injury+DFO groups were all 0 points, indicating the effectiveness of the SCI model of the rats. The score increased with time, however, at 7 days after SCI, the BBB score between the Injury and Injury+DFO groups was statistically significant. At 8 weeks after SCI, the BBB score of group Injury was 14, and Injury+DFO group 18. showed that DFO had a significant recovery of the motor function after SCI in rats. [Conclusion] DFO can significantly reduce the part of the iron ions containing SCI. Quantity, reduce the expression of SCI local TNF alpha, IL1- beta and Caspase-3, protect local original structure cells, reduce cell apoptosis, restore the motor function of the hind limbs of rats, promote the repair of SCI, provide new drug treatment options for the treatment of SCI, and provide experimental basis for the clinical repair of SCI.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

【参考文献】