膜粘连蛋白Ⅱ在颅脑创伤后急性期与慢性期中的神经保护作用及机制研究

发布时间：2018-04-16 01:28

本文选题：创伤性颅脑损伤 + 膜粘连蛋白Ⅱ　；参考：《重庆医科大学》2015年博士论文

【摘要】：第一部分膜粘连蛋白Ⅱ通过保护血脑屏障对小鼠颅脑创伤后早期神经功能恢复的影响目的：评估外源性重组A2对创伤性颅脑损伤后早期神经功能预后以及血脑屏障的影响,探讨A2作为脑创伤治疗靶点的可能性。方法：实验一：内源性A2蛋白在CCI后不同时间点和不同细胞内表达的变化在成年雄性小鼠上建立精确皮质打击模型,检测伤前与伤后不同时间点(6小时,1天,3天,5天,7天,14天,21天)伤侧半球脑组织A2蛋白的表达；用CD31、GFAP、NeuN分别标记内皮细胞、星形胶质细胞和神经元,再与A2抗体行双重染色检测伤后A2在不同细胞类型中表达的变化情况。实验二：外源性A2蛋白对CCI后BBB通透性、脑水肿以及海马神经元存活的影响体外培养内皮细胞并建立损伤模型,检测rA2对伤后内皮细胞电阻、内皮细胞屏障通透性和内皮细胞活力的影响；小鼠CCI伤后通过静脉注射伊文思蓝染料并测定半球染料含量,检测rA2对BBB通透性的影响；通过干湿重法测定伤后大脑半球组织含水量,检测rA2对伤后脑水肿的影响；免疫组织荧光对CCI后海马区神经元进行标记并定量,检测rA2伤后海马神经元存活的影响；给予离体缺氧处理的神经元中加入内皮细胞条件培养基,检测rA2通过内皮细胞对受损神经元的细胞活力影响。实验三：外源性A2蛋白对CCI后早期神经功能恢复和伤灶体积的影响通过NSS评分、Wire-gripping实验分别对CCI伤后小鼠进行检测,观察rA2对颅脑创伤后早期神经功能恢复的影响；对CCI伤后小鼠的组织切片进行HE染色,观察rA2对颅脑创伤后伤灶体积的影响。实验四：外源性A2蛋白对脑损伤后BBB保护作用的机制通过明胶酶谱法,检测rA2对颅脑创伤后脑组织MMP-9活性的影响；通过对伤侧脑组织中蛋白质定量,检测rA2对颅脑创伤后VEGF、 Occludin、Claudin-5和ZO-1表达的影响；建立体外内皮细胞损伤模型,检测rA2对内皮细胞应力纤维形成的影响。结果：实验一：从伤后3天起,A2表达明显增高(P0.05),并在7天时达到高峰(P0.05)。此后,蛋白表达逐渐下降,21天时恢复到基值。对比伤前与伤后免疫组织荧光结果,伤前内皮细胞几乎不表达A2,伤后在伤侧部分内皮细胞表达A2；伤前星形胶质细胞部分表达A2,伤后双侧胶质细胞均有活化,且活化的胶质细胞都能表达A2；神经元细胞在伤前已有A2表达,伤后未见明显改变。实验二：在内皮细胞损伤后rA2能显著提高内皮细胞电阻(P0.05)和细胞活力(P0.05),并降低FITC-dextran通过内皮细胞屏障的数量(P0.05)；通过在伤后不同时间点(2小时,4小时,6小时)将不同剂量rA2 (0.75mg/kg, 1mg/kg,1.5mg/kg)通过尾静脉注入小鼠体内,均能显著降低伤后脑组织EB渗出量(P0.05)；给予rA2不能显著改变伤后脑组织水肿状况(P0.05)；rA2能显著减少伤后7天CA1和CA3区神经元丢失,减轻海马区结构破坏(P0.05)；给予rA2的内皮细胞条件培养基能显著提高缺氧损伤神经元的活力(P0.05)。实验三：NSS评分结果证实rA2能显著提高伤后7天内运动相关行为学表现(P0.05),Wire-gripping实验中rA2组小鼠的表现与对照组无显著差别(P0.05)；HE染色证实给予rA2后伤灶体积无明显改变(P0.05)。实验四：rA2对伤后7天小鼠脑组织MMP-9活性无显著影响(P0.05)；rA2对伤后7天小鼠脑组织中VEGF、Occludin和Claudin-5表达无显著影响(P0.05)；rA2能显著增加伤后7天小鼠脑组织中ZO-1表达(P0.05)；给予rA2后受损的内皮细胞内的应力纤维数量显著降低(P0.05)。结论：在创伤性颅脑损伤早期,rA2能提高内皮细胞ZO-1合成,减少细胞内应力纤维数量,从而稳定内皮细胞间紧密连接,减少BBB通透性。这种BBB的修复作用能保护神经组织,从而减少神经元凋亡,促进伤后早期神经功能恢复。第二部分膜粘连蛋白Ⅱ通过血管再生途径对小鼠颅脑创伤后远期神经功能恢复的影响目的：评估外源性重组A2对创伤性颅脑损伤后长期神经功能预后的影响,同时探讨A2促进神经功能修复的机制。方法：实验一：外源性A2蛋白对颅脑创伤后远期神经功能恢复和伤灶体积的影响通过NSS评分、转棒实验和水迷宫实验分别将CCI伤后小鼠进行检测,观察rA2对颅脑创伤后神经功能恢复的影响；对CCI伤后小鼠的组织切片进行HE染色,观察rA2对颅脑创伤后伤灶体积的影响。实验二：外源性A2蛋白对颅脑创伤后血管再生的影响及其机制离体培养内皮细胞,检测rA2对内皮细胞增殖、管腔结构形成和运动迁移的影响；通过CD31/Brdu标记新生内皮细胞,检测rA2对颅脑创伤后内皮细胞增殖的影响；通过静脉注射Lectin,检测rA2对颅脑创伤后功能性血管密度的影响；分离伤后皮质微血管并进行原位酶活性检测,比较rA2对颅脑创伤后血管血纤维蛋白溶酶活性的影响。实验三：外源性A2蛋白对颅脑创伤后神经再生的影响及其机制通过标记Nestin/Brdu标记增殖的神经干细胞,检测rA2对颅脑创伤后神经干细胞增殖的影响；通过NeuN/Brdu标记新生的神经元,检测rA2对颅脑创伤后神经再生的影响；通过PSA-NCAM/Brdu标记新生的成神经细胞,检测rA2对颅脑创伤后成神经细胞迁移的影响；通过对伤侧脑组织中蛋白质定量,检测rA2对颅脑创伤后神经营养因子的影响；分离伤后皮质微血管并提取mRNA,检测rA2对颅脑创伤后血管内皮内神经营养因子基因转录水平的影响。结果：实验一：NSS评分、转棒实验结果证实rA2能显著提高伤后28天内运动相关行为学表现(P0.05),水迷宫实验中rA2组小鼠的空间记忆能力明显优于对照组小鼠(P0.05)；HE染色证实给予rA2后伤灶体积无明显改变(P0.05)。实验二：离体培养条件下,rA2能显著促进内皮细胞增殖、管腔结构形成与运动迁移能力(P0.05)；rA2能显著增加CCI伤后7天内海马和伤灶周围内皮细胞增殖(P0.05)；伤后28天时,rA2组伤灶周围皮质功能性血管密度显著高于对照组(P0.05),但海马区血管网密度无明显变化(P0.05)；rA2提高伤后7天皮质微血管内血纤维蛋白溶酶活性。实验三：rA2对伤后早期SGZ和SVZ区域的神经干细胞分裂增殖无显著作用(P0.05),但是却能增加后期伤灶区域新生神经元的数量(P0.05)；rA2增加了伤后7天内迁移至伤灶周围以及海马齿状回的新生神经元数量(P0.05)；rA2增加伤侧脑组织中BDNF、VEGF的含量；分离伤后7天时小鼠脑组织皮质微血管,rA2组微血管内BDNF、 VEGF的mRNA转录水平显著对照组(P0.05)。结论：在创伤性颅脑损伤后,rA2能提高微血管内血纤维蛋白溶酶活性,从而促进伤后血管再生。同时,增生活跃的内皮细胞能分泌更多的神经营养因子,这些因子能吸引成神经细胞沿着血管网走行向受伤区域迁移,到达后发育为成熟的神经元,从而参与伤后的神经修复重建,最终促进伤后长期神经功能恢复。
[Abstract]:The first part of the membrane adhesion protein by protecting blood brain barrier effect on early recovery of nerve function after traumatic brain injury in mice Objective: To evaluate the effect of exogenous A2 on early prognosis of neurological function and blood brain barrier after traumatic brain injury, brain trauma of A2 as the target of treatment possibilities. Methods: experiment 1: the change of endogenous A2 expression of CCI in different time points after different cells and establish accurate cortical hit model in adult male mice, detected before and after injured at different time points (6 hours, 1 days, 3 days, 5 days, 7 days, 14 days, 21 days) the expression of A2 protein in brain tissue of the injured hemisphere with CD31, GFAP, NeuN; were labeled endothelial cells, astrocytes and neurons, changes with A2 antibody double staining detection after injury, the expression of A2 in different cell types. Experiment two: exogenous A2 protein on CCI BBB permeability Of brain edema and effects of hippocampal neurons in vitro cultured endothelial cells and establish the injury model, detection of rA2 on endothelial cell injury resistance, effect of endothelial barrier permeability and endothelial cell viability; CCI mice after injury by intravenous injection of Evans blue dye and measuring hemisphere dye content, to study the effect of rA2 on the permeability of BBB; Determination of injury by dry wet method after the brain tissue water content, to study the effect of rA2 on brain edema after cerebral injury; immunohistochemistry of CCI of hippocampus neurons were labeled and quantitative detection effect of hippocampal neural Yuan Cunhuo rA2 after injury; give away from the medium with endothelial cell conditioned body hypoxia treated neurons, by detection of rA2 endothelial cells on damaged neurons cell viability. Experiment three: the effect of exogenous A2 protein on CCI early after the recovery of neurological function and infarct volume through injury The NSS score, Wire-gripping test were used to detect CCI after injury in mice, the effect of rA2 on the effect of early recovery of nerve function after traumatic brain injury; the staining of CCI after injury in mice tissues were stained with HE, the effect of rA2 on traumatic brain injury after traumatic volume. Experiment four: the mechanism of exogenous A2 protein on brain injury the protective effect of BBB by gelatin zymography, to study the effect of rA2 on the activity of MMP-9 in brain tissue after traumatic brain injury; the quantitative protein injured side brain tissue, detection of rA2 for traumatic brain injury after VEGF, Occludin, expression of Claudin-5 and ZO-1; in vitro endothelial cell injury model, detect the effect of rA2 stress on fiber formation endothelial cells. Results: experiment one: from the 3 day after injury, the expression of A2 increased significantly (P0.05), and reached the peak at day 7 (P0.05). Since then, the expression decreased gradually, the 21 day return to the base value of contrast. Before and after injured immunohistofluorescence results before the injury of endothelial cells, almost no expression of A2 after injury in the injured side, part of the expression of A2 of endothelial cells; injury of astrocytes partially expressed A2, had bilateral activation of glial cells after injury, and the activation of glial cells can express A2; neurons expression in existing A2 injury before, after the injury did not change significantly. Experiment two: rA2 can significantly improve the resistance in endothelial cells after injury of endothelial cell (P0.05) and cell viability (P0.05), and reduce the number of endothelial cells through the FITC-dextran barrier (P0.05); the same time point not after injury (2 hours, 4 hours, 6 hours) different doses of rA2 (0.75mg/kg, 1mg/kg, 1.5mg/kg) were injected into mice by tail vein, can significantly reduce the injury of brain tissue exudation of EB (P0.05); rA2 did not significantly alter the injury status of brain edema (P0.05); rA2 can significantly reduce the injury after 7 days CA1 and CA3 area neuron loss, reduce the damage zone structure of hippocampus (P0.05); rA2 endothelial cell conditioned medium can significantly improve the hypoxic neuronal activity (P0.05). Experiment three: NSS score results confirmed that rA2 can significantly improve the exercise after injury within 7 days of behavioral performance (P0.05), rA2 group the mice in the Wire-gripping experiment showed no significant difference with the control group (P0.05); HE staining confirmed after giving rA2 injury. The volume did not change significantly (P0.05). Experiment four: rA2 had no significant effect on the 7 day after injury in mice brain tissue MMP-9 activity (P0.05); rA2 VEGF on the 7 day after injury in mice brain Occludin, and the expression of Claudin-5 had no significant effect (P0.05); rA2 ZO-1 expression was significantly increased 7 days after injury in mouse brain tissue (P0.05); the number of stress fibers after giving rA2 damaged endothelial cells decreased significantly (P0.05). Conclusion: in the early stage of traumatic brain injury, RA2 can improve the endothelial cell ZO-1 synthesis, reduce the number of cell stress fibers, thereby stabilizing endothelial tight junctions between cells, reduce the permeability of BBB. This repair BBB can protect the nerve tissue, thereby reducing neuronal apoptosis, promote early recovery of nerve function after injury. The second part film adhesion protein II through vascular regeneration effect on long-term recovery the nerve function after traumatic brain injury in mice Objective: To evaluate the effects of exogenous recombinant A2 on traumatic brain injury after long-term neurological outcome, and explore the mechanism of A2 promoting the recovery of neural function. Methods: experiment one: effects of exogenous A2 protein on long-term neurological functional recovery after traumatic brain injury and traumatic volume by NSS score, turn bar test and water maze test respectively after injury CCI mice were detected, the effect of rA2 on the recovery of nerve function after traumatic brain injury of CCI after injury; Mouse tissue sections were stained with HE, the effect of rA2 on traumatic brain injury after traumatic volume. Experiment two: the effect of exogenous A2 protein on vascular regeneration after traumatic brain injury and its mechanism in vitro cultured endothelial cells, detection of rA2 on endothelial cell proliferation, migration and lumen formation influence movement; through endothelial CD31/Brdu markers cell detection effect of rA2 on proliferation of endothelial cells after traumatic brain injury; by intravenous injection of Lectin, detection of rA2 effects on functional vascular density after traumatic brain injury; for in situ detection of enzyme activity and cortical microvascular separation after injury, to compare the effects of rA2 on vascular plasmin activity after traumatic brain injury: experiment three. Exogenous A2 protein effect on nerve regeneration after traumatic brain injury and its mechanism by marking the proliferation of neural stem cell marker Nestin/Brdu, rA2 detection of traumatic brain injury neural stem cells Effect of cell proliferation by NeuN/Brdu markers; new neurons, detection of rA2 effect on nerve regeneration after traumatic brain injury; nerve cells through PSA-NCAM/Brdu labeled newborn, detect the influence of rA2 on craniocerebral trauma after nerve cell migration; through the quantification of protein in the brain tissue of the injured side, detect the effect of rA2 on neurotrophic factor in the brain trauma; injury of cortical microvascular separation and extraction of mRNA, to study the effect of rA2 on neurotrophic factor gene expression after traumatic brain injury in vascular endothelial cells. Results: experiment one: NSS score, rotarod test results showed that rA2 showed movement within 28 days after the injury related behavior increased significantly (P0.05), space the memory ability of rA2 mice in the water maze test was better than the control group (P0.05); HE staining confirmed after giving rA2 injury. The volume did not change significantly (P0.05). Experiment two: in vitro culture. Under the condition, rA2 can significantly promote endothelial cell proliferation, lumen formation and migration ability (P0.05); rA2 within 7 days of the hippocampus and wound around the focus of endothelial cell proliferation significantly increased CCI after injury (P0.05); 28 days after injury, injury group rA2 cerebral cortex functional vascular density was significantly higher than that of the control group (P0.05), but in hippocampus of vascular density did not change significantly (P0.05); rA2 increased 7 days after injury of cortical microvascular plasmin activity. Experiment three: rA2 had no significant effect on SGZ and SVZ cells in the early region of neural stem proliferation after injury (P0.05), but the number can increase in post traumatic area of new neurons (P0.05); rA2 increased 7 days after injury to the wound around the focus and migration of newborn neurons in the hippocampal dentate gyrus (P0.05); the number of rA2 increased BDNF injury in the brain tissues, the content of VEGF; the separation of 7 days after the injury in brain tissue of small rat cortical microvascular, Group rA2 microvascular BDNF, VEGF transcription of mRNA significantly in control group (P0.05). Conclusion: in traumatic brain injury, rA2 can improve the microvascular plasmin activity, thereby promoting vascular regeneration after injury. At the same time, hyperplasia of endothelial cells can secrete neurotrophic factors more, these factors can attract into neural cells along the vascular network to go after the arrival of the injured area migration, the formation of mature neurons, and thus participate in the repair and reconstruction of nerve injury, and ultimately promote the recovery of nerve function after long-term injury.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.15

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