芬戈莫德对脊髓损伤后神经功能修复及体外神经干细胞影响的研究

发布时间：2018-06-06 20:47

本文选题：免疫抑制剂 + 芬戈莫德　；参考：《重庆医科大学》2015年硕士论文

【摘要】：目的通过体内实验观察FTY720对大鼠急性脊髓损伤后神经功能的修复作用和血-脊髓屏障功能的影响,探讨相关的作用机制；同时初步观察FTY720-P对体外NSCs增殖、迁移和分化的影响,为后续实验和临床脊髓损伤治疗提供实验依据和新思路。方法 1.制备SD大鼠脊髓半横切损伤模型并随机分为四组：正常对照组(NG组)、假手术组(SO组)、损伤对照组(HS组)、芬戈莫德治疗组(FTY720组)。分别于损伤后1天、3天、7天、14天、28天对各组大鼠进行BBB运动功能评分、水平网格实验观察损伤侧后肢运动和放置功能；神经诱发电位实验观察损伤侧神经束传导功能；HE染色观察损伤周围组织病理形态变化以系统评价FTY720对神经功能修复作用的影响；最后通过伊文思蓝渗透性实验观察FTY720对大鼠脊髓损伤后血-脊髓屏障功能的影响,探讨其可能的作用机制。2.采用孕14.5-16.5天的SD大鼠胎鼠进行NSCs的体外分离培养,并利用细胞免疫荧光化学染色法对NSCs相关生物学特性进行鉴定；观察体外培养的NSCs在不同浓度的磷酸化形式FTY720-P(0nM、 1nM、10nM、100nM)作用下其增殖率、迁移能力及分化方向的改变。结果 1.半横切损伤后,HS组和FTY720组大鼠损伤侧脊髓神经功能下降,至28天时仍未恢复至NG组和SO组水平。行为学检测和神经电生理检测结果提示,FTY720组大鼠运动功能恢复速度比HS组更快；损伤7、14、28天时,两组大鼠BBB评分和SEP P1波潜伏期检测差异有统计学意义(P0.05)；损伤14、28天时,两组大鼠水平网格检测和MEP Nl波潜伏期检测差异有统计学意义(P0.05)。组织学检测结果显示,各时间点NG组和SO组大鼠脊髓结构均完整；损伤14天时,FTY720组脊髓损伤处慢性炎症细胞浸润,胶质化反应程度小于HS组；损伤28天时,FTY720组损伤处空洞残存面积小于HS组,再生纤维排列较HS组有序。2.血-脊髓屏障渗透性检测结果发现,损伤后7天内,HS组和FTY720组大鼠血-脊髓屏障EB渗出量较NG组和SO组明显增加；各时间点FTY720组EB渗出面积均小于HS组(P0.05),其中损伤3天时差异最为显著。3.体外NSCs成功提取、分离培养并鉴定。4.在未添加bFGF的培养基中,不同浓度FTY720-P处理后的各组NSCs增殖率较对照组差异不明显。在添加有bFGF的培养基中,NSCs增殖率明显增加,其中10nM、100nM组细胞增殖率增加显著,与对照组比较有统计学差异(P0.05)。5.利用细胞免疫荧光染色对体外诱导分化的NSCs行β-tublinⅢ和GFAP双标鉴定发现,1nM、10nM、100nM浓度的FTY720-P对β-tublinⅢ表达阳性的神经元和GFAP表达阳性的星型胶质细胞分化比例没有显著的影响。但在分化出的星型胶质细胞中,随着FTY720 -P浓度的增加,原浆型星型胶质细胞的比例上升。6.利用细胞小室迁移和结晶紫染色实验观察FTY720-P对NSCs迁移能力的影响发现,随FTY720-P浓度增加,NSCs迁移数量增加,10nM、100nM组NSCs迁移数量增加明显,与对照组比较差异显著(P0.05)。结论 FTY720能显著减少脊髓损伤后急性期血-脊髓屏障渗透性,有效促进急性期后神经功能的恢复；体外实验证实FTY720-P在一定剂量下能与bFGF协同作用,促进NSCs增殖；对NSCs产生化学趋化作用,促进NSCs的迁移；同时,FTY720-P还能影响分化方向,提高分化胶质细胞中原浆型星型胶质细胞的比例。证实FTY720-P能对体外NSCs生物学特性有一定调节作用。
[Abstract]:Objective To observe the effect of FTY720 on the repair of nerve function and the function of blood spinal cord barrier after acute spinal cord injury in rats, and to explore the effect of FTY720-P on the proliferation, migration and differentiation of NSCs in vitro, and provide the experimental basis and new thought for the treatment of spinal cord injury in the follow-up experiment and clinical practice. Method 1. the spinal cord hemisection damage model of SD rats was randomly divided into four groups: normal control group (group NG), sham operation group (group SO), injury control group (group HS) and Finn Gomo de group (group FTY720). The BBB motor function scores of rats were scored at 1 days, 3 days, 7 days, 14 days and 28 days after injury, and the injured side was observed by horizontal grid experiment. The posterior limb movement and placement function; the nerve evoked potential test was used to observe the nerve tract conduction function of the injured side; HE staining was used to observe the pathological changes of tissue around the injury in order to evaluate the effect of FTY720 on the function of nerve function repair. Finally, the blood spinal barrier function after spinal cord injury in rats was observed by the Evans blue permeability test. The possible mechanism of effect was to explore its possible mechanism of action.2. using SD rats pregnant with pregnant 14.5-16.5 days to isolate and culture NSCs in vitro, and to identify the biological characteristics of NSCs by cell immunofluorescence chemical staining, and to observe the effect of NSCs in the form of phosphorylation of FTY720-P (0nM, 1nM, 10nM, 100nM) in different concentrations in vitro. The proliferation rate, migration ability and differentiation direction were changed. Results after 1. half transection injury, the spinal nerve function of the injured side of the HS group and FTY720 group decreased to the level of the NG group and the SO group at 28 days. The results of behavior detection and electrophysiological detection suggested that the recovery speed of motor function in FTY720 group was faster than that of the HS group; 7,14 was damaged in 7,14. At 28 days, there was significant difference in BBB score and SEP P1 wave latency detection in two groups of rats (P0.05). At 14,28 days of injury, there was a significant difference between the level grid detection and the MEP Nl wave latency detection in two groups of rats (P0.05). The histological examination showed that the spinal structure of the NG group and the SO group of the rats at all time points were all intact, and the injury was 14 days, FT. In group Y720, the chronic inflammatory cell infiltration and the degree of glial reaction were less than that of the HS group. When the injury was 28 days, the residual cavity area in the FTY720 group was less than that of the group HS, and the regenerated fiber was found to be more than the HS group in order.2. blood spinal barrier permeability test. The EB exudation of the blood spinal cord barrier in the HS group and the FTY720 group was more than the NG group within the HS group and the FTY720 group. The area of EB exudation in group FTY720 was less than that of group HS (P0.05) at all time points, and the difference was the most significant of.3. in 3 days after 3 days, and the difference of.4. in the medium without bFGF was isolated and identified, and the NSCs colonization rate of each group of FTY720-P treated with FTY720-P was not obvious. In the medium, the proliferation rate of NSCs increased significantly, in which the proliferation rate of 10nM and 100nM increased significantly. Compared with the control group, there was a statistically significant difference (P0.05).5. using cell immunofluorescence staining to identify NSCs in vitro induced by beta -tublin III and GFAP In the differentiated astrocytes, the proportion of FTY720 -P increased with the increase of -P concentration in the astrocytes. The effect of FTY720-P on the migration ability of NSCs was observed by the cell migration and crystal violet staining experiments, which were observed with FTY720. The increase of -P concentration, the number of NSCs migration increased, the number of NSCs migration in group 100nM increased significantly in 10nM and 100nM group, and the difference was significant (P0.05). Conclusion FTY720 can significantly reduce the permeability of blood spinal cord barrier after spinal cord injury, effectively promote the recovery of nerve function after acute stage, and in vitro experiments confirm that FTY720-P can be in a certain dose and bFGF under a certain dose. Synergistic action promotes the proliferation of NSCs, produces chemical chemotaxis to NSCs and promotes the migration of NSCs. At the same time, FTY720-P can also influence the direction of differentiation and increase the proportion of astrocytes in glial cells of differentiated glial cells. It is confirmed that FTY720-P can have a certain regulating effect on the biological characteristics of NSCs in vitro.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

【相似文献】