發(fā)布時間：2018-06-06 20:47

  本文選題：免疫抑制劑 + 芬戈莫德　； 參考：《重慶醫(yī)科大學》2015年碩士論文

【摘要】：目的 通過體內實驗觀察FTY720對大鼠急性脊髓損傷后神經功能的修復作用和血-脊髓屏障功能的影響,探討相關的作用機制；同時初步觀察FTY720-P對體外NSCs增殖、遷移和分化的影響,為后續(xù)實驗和臨床脊髓損傷治療提供實驗依據和新思路。方法 1.制備SD大鼠脊髓半橫切損傷模型并隨機分為四組：正常對照組(NG組)、假手術組(SO組)、損傷對照組(HS組)、芬戈莫德治療組(FTY720組)。分別于損傷后1天、3天、7天、14天、28天對各組大鼠進行BBB運動功能評分、水平網格實驗觀察損傷側后肢運動和放置功能；神經誘發(fā)電位實驗觀察損傷側神經束傳導功能；HE染色觀察損傷周圍組織病理形態(tài)變化以系統(tǒng)評價FTY720對神經功能修復作用的影響；最后通過伊文思藍滲透性實驗觀察FTY720對大鼠脊髓損傷后血-脊髓屏障功能的影響,探討其可能的作用機制。2.采用孕14.5-16.5天的SD大鼠胎鼠進行NSCs的體外分離培養(yǎng),并利用細胞免疫熒光化學染色法對NSCs相關生物學特性進行鑒定；觀察體外培養(yǎng)的NSCs在不同濃度的磷酸化形式FTY720-P(0nM、 1nM、10nM、100nM)作用下其增殖率、遷移能力及分化方向的改變。結果 1.半橫切損傷后,HS組和FTY720組大鼠損傷側脊髓神經功能下降,至28天時仍未恢復至NG組和SO組水平。行為學檢測和神經電生理檢測結果提示,FTY720組大鼠運動功能恢復速度比HS組更快；損傷7、14、28天時,兩組大鼠BBB評分和SEP P1波潛伏期檢測差異有統(tǒng)計學意義(P0.05)；損傷14、28天時,兩組大鼠水平網格檢測和MEP Nl波潛伏期檢測差異有統(tǒng)計學意義(P0.05)。組織學檢測結果顯示,各時間點NG組和SO組大鼠脊髓結構均完整；損傷14天時,FTY720組脊髓損傷處慢性炎癥細胞浸潤,膠質化反應程度小于HS組；損傷28天時,FTY720組損傷處空洞殘存面積小于HS組,再生纖維排列較HS組有序。2.血-脊髓屏障滲透性檢測結果發(fā)現,損傷后7天內,HS組和FTY720組大鼠血-脊髓屏障EB滲出量較NG組和SO組明顯增加；各時間點FTY720組EB滲出面積均小于HS組(P0.05),其中損傷3天時差異最為顯著。3.體外NSCs成功提取、分離培養(yǎng)并鑒定。4.在未添加bFGF的培養(yǎng)基中,不同濃度FTY720-P處理后的各組NSCs增殖率較對照組差異不明顯。在添加有bFGF的培養(yǎng)基中,NSCs增殖率明顯增加,其中10nM、100nM組細胞增殖率增加顯著,與對照組比較有統(tǒng)計學差異(P0.05)。5.利用細胞免疫熒光染色對體外誘導分化的NSCs行β-tublinⅢ和GFAP雙標鑒定發(fā)現,1nM、10nM、100nM濃度的FTY720-P對β-tublinⅢ表達陽性的神經元和GFAP表達陽性的星型膠質細胞分化比例沒有顯著的影響。但在分化出的星型膠質細胞中,隨著FTY720 -P濃度的增加,原漿型星型膠質細胞的比例上升。6.利用細胞小室遷移和結晶紫染色實驗觀察FTY720-P對NSCs遷移能力的影響發(fā)現,隨FTY720-P濃度增加,NSCs遷移數量增加,10nM、100nM組NSCs遷移數量增加明顯,與對照組比較差異顯著(P0.05)。結論 FTY720能顯著減少脊髓損傷后急性期血-脊髓屏障滲透性,有效促進急性期后神經功能的恢復；體外實驗證實FTY720-P在一定劑量下能與bFGF協(xié)同作用,促進NSCs增殖；對NSCs產生化學趨化作用,促進NSCs的遷移；同時,FTY720-P還能影響分化方向,提高分化膠質細胞中原漿型星型膠質細胞的比例。證實FTY720-P能對體外NSCs生物學特性有一定調節(jié)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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R651.2

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