miR-21-5p对颅脑创伤后神经-血管单元修复的影响及作用机制研究

发布时间：2018-07-07 13:47

本文选题：创伤性脑损伤 + miR-21-5p　；参考：《天津医科大学》2015年博士论文

【摘要】：研究目的:创伤性颅脑损伤(TBI)致残、致死率高,其所致的继发性脑损伤与TBI预后密切相关。神经-血管单元(NVU)是由神经元、神经胶质细胞、血管内皮细胞及细胞外基质等构成的功能单元,通过其中各种成分的相互作用维持脑组织内环境稳态。促进TBI后NVU损伤修复是治疗继发性脑损伤、救治TBI的关键。mi RNAs在TBI领域中的研究报道尚少。我所在课题组的前期研究发现,TBI大鼠创伤侧大脑皮层mi R-21-5p表达量较伤前明显升高。此外,初步证实mi R-21-5p表达水平的升高可以减轻脑组织细胞凋亡,并改善TBI大鼠的神经功能预后。本实验将通过动物实验,进一步阐明mi R-21-5p在NVU损伤修复中的作用及机制,为探索TBI后mi RNAs治疗的新策略提供理论依据。方法:1.应用q RT-PCR及mi RNA原位杂交+免疫荧光双染技术,检测TBI后mi R-21-5p在创伤灶脑组织的表达变化,及其在神经元、星形胶质细胞、小胶质细胞及脑微血管内皮细胞中的原位表达变化。2.通过改进的神经功能评分及水迷宫实验,评价TBI大鼠神经功能;应用TUNEL法细胞凋亡检测,评价mi R-21-5p对脑组织细胞凋亡的影响;应用免疫荧光法检测脑微血管密度,评价mi R-21-5p对脑组织血管生成修复的影响;通过脑组织干湿重检测、血脑屏障(BBB)Evans Blue渗漏量检测及紧密连接蛋白(Occludin及Claudin-5)定量检测,评价mi R-21-5p对BBB通透性的影响。3.应用Western Blot和q RT-PCR技术,检测mi R-21-5p可能的作用靶点PTEN、AKT信号通路及其下游蛋白(凋亡相关蛋白Bax、Bcl-2、Caspase-3;血管生成及稳定性相关因子Ang-1、Tie-2)的表达变化,探讨mi R-21-5p影响细胞凋亡、血管生成修复及BBB通透性的相关机制。结果:1.(1)TBI后,创伤灶脑组织mi R-21-5p表达量自伤后6h起逐渐升高,于伤后3d达峰,随后逐渐下降,于伤后14d降低至假手术组大鼠水平;且mi R-21-5p在神经元、星形胶质细胞、小胶质细胞、脑微血管内皮细胞(BMVECs)中的原位表达水平均有不同程度的升高。(2)在TBI前后,mi R-21-5p在脑神经组织中均主要表达于神经元及星形胶质细胞。(3)通过侧脑室注射脂质体携带的mi R-21-5p agomir/antagomir,可以上调/下调创伤灶脑组织于伤后6h、1d及3d的mi R-21-5p表达水平,且同时上调/下调mi R-21-5p在以上细胞中的原位表达水平。2.(1)TBI后,脑组织mi R-21-5p表达水平的升高可以改善神经功能预后。(2)mi R-21-5p的表达可以抑制脑组织细胞凋亡。(3)mi R-21-5p的表达可以促进脑组织血管生成修复。(4)mi R-21-5p的表达可以减轻BBB渗漏。3.(1)TBI后,脑组织mi R-21-5p的表达量可以影响凋亡相关蛋白的表达水平。(2)mi R-21-5p可以在转录后水平靶向抑制脑组织PTEN基因的表达,并促进Akt磷酸化。(3)mi R-21-5p的表达可以促进脑组织血管生成及稳定性相关因子Ang-1及Tie-2的表达。结论:1.TBI后,创伤灶脑组织mi R-21-5p表达水平升高,且在神经元、星形胶质细胞、小胶质细胞、脑微血管内皮细胞中的原位表达水平均有不同程度的升高。2.TBI后,mi R-21-5p通过在转录后水平抑制靶基因PTEN的表达,激活Akt通路,从而调控下游凋亡相关蛋白的表达,产生抑制脑组织细胞凋亡的作用。3.TBI后,BMVECs表达的mi R-21-5p通过激活Ang-1/Tie-2通路,产生促进脑组织血管生成修复、减轻BBB渗漏的作用。4.TBI后,mi R-21-5p可以通过上述作用,保护神经-血管单元,减轻继发性脑损伤,从而改善TBI预后。5.mi R-21-5p是TBI后继发性脑损伤的潜在治疗靶点。
[Abstract]:Objective: traumatic brain injury (TBI) is disabled and has a high mortality rate. The secondary brain damage caused by it is closely related to the prognosis of TBI. The neurovascular unit (NVU) is a functional unit consisting of neurons, glial cells, vascular endothelial cells and extracellular matrix, and maintains the internal environment of the brain through the interaction of various components. NVU damage repair after TBI is the treatment of secondary brain injury, and the key.Mi RNAs in the treatment of TBI is rarely reported in the field of TBI. The earlier study in my group found that the R-21-5p expression of MI in the cerebral cortex of the TBI rats was significantly higher than that before the injury. Furthermore, it was preliminarily confirmed that the increase in the expression level of MI R-21-5p could be reduced. The apoptosis of brain tissue and the prognosis of neural function in TBI rats will be improved. Through animal experiments, this experiment will further elucidate the role and mechanism of MI R-21-5p in the repair of NVU damage, and provide a theoretical basis for exploring new strategies for MI RNAs treatment after TBI. Methods: 1., Q RT-PCR and Mi RNA in situ hybridization + immunofluorescence double staining technique was used to detect TBI after TBI. The changes in the expression of MI R-21-5p in the brain tissue of the trauma, and in situ expression changes in neurons, astrocytes, microglia and cerebral microvascular endothelial cells..2. was evaluated by improved neural function score and water maze test to evaluate the neural function of TBI rats. The TUNEL method of apoptosis was used to evaluate the Mi R-21-5p on the brain tissue. The effect of apoptosis, detection of cerebral microvascular density by immunofluorescence, and evaluation of the effect of MI R-21-5p on angiogenesis and repair of brain tissue, quantitative detection of blood brain barrier (BBB) Evans Blue leakage and quantitative detection of close connexin (Occludin and Claudin-5) by brain tissue dry and wet weight, and to evaluate the.3. application of MI R-21-5p to BBB permeability Western Blot and Q RT-PCR techniques are used to detect the possible targets of MI R-21-5p, PTEN, AKT signaling pathway and its downstream proteins (Bax, Bcl-2, Caspase-3, angiogenesis and stability related factors). After 1. (1) TBI, the expression of MI R-21-5p in the traumatic brain tissue increased gradually after the injury, and the 3D reached the peak after injury, and then decreased gradually. The 14d decreased to the level of the rat in the sham operation group after injury, and the expression level of MI R-21-5p in the neurons, astrocytes, microglia and intracerebral microvascular skin cells (BMVECs) were in varying degrees. (2) before and after TBI, MI R-21-5p was mainly expressed in neurons and astrocytes in the brain nerve tissue. (3) the MI R-21-5p agomir/antagomir carried by liposomes was injected into the lateral ventricle. The MI R-21-5p expression of 6h, 1D and 3D in the traumatic brain tissue could be up-regulated and downregulated at the same time. After the expression level of.2. (1) TBI in the cell, the elevation of the expression level of MI R-21-5p in the brain can improve the prognosis of neural function. (2) the expression of MI R-21-5p can inhibit the apoptosis of the brain tissue. (3) the expression of MI R-21-5p can promote the angiogenesis and repair of the brain tissue. (4) the expression of MI R-21-5p can reduce BBB leakage.3. (1) The expression of 21-5p can affect the expression level of apoptosis related proteins. (2) mi R-21-5p can inhibit the expression of PTEN gene in the brain tissue at post transcriptional level and promote Akt phosphorylation. (3) the expression of MI R-21-5p can promote the expression of Ang-1 and Tie-2 in the angiogenesis and stability related factors of brain tissue. Conclusion: after 1.TBI, the brain tissue of the traumatic brain tissue The expression level of MI R-21-5p increased and the expression level of in situ expression in neurons, astrocytes, microglia and cerebral microvascular endothelial cells increased to varying degrees of.2.TBI. Mi R-21-5p inhibited the expression of target gene PTEN and activated the Akt pathway through the post transcriptional level, thus regulating the expression of downstream apoptosis related proteins and producing inhibition of the expression of apoptosis related proteins. After the effect of.3.TBI on the apoptosis of brain tissue cells, the MI R-21-5p expressed by BMVECs activates the Ang-1/Tie-2 pathway to promote the angiogenesis and repair of the brain tissue, and reduces the effect of BBB leakage on.4.TBI. Mi R-21-5p can protect the nerve and vascular units and reduce the subsequent brain damage. Thus, the.5.mi R-21-5p is the prognosis of TBI. The.5.mi R-21-5p is the prognosis of TBI. Potential therapeutic targets for secondary secondary brain injury.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.15

【共引文献】