Treg输注移植通过抑制神经炎症对SAH小鼠脑组织的保护作用研究

发布时间：2019-05-20 10:14

【摘要】：目的：蛛网膜下腔出血(subarachnoid hemorrhage, SAH)是常见的脑组织出血性病变,因其致死致残率高,且存活者多预后不良,造成患者生活质量下降。研究证实SAH患者脑血管痉挛(cerebralvasospasm, CVS)和早期脑损伤(Early brain injury, EBI)的严重程度,与不良预后相关。因此,非常有必要研制新型药物,控制SAH后早期炎性免疫反应,减轻EBI和CVS的致病性,减轻患者的痛苦。有研究证实,调节性T细胞(CD4+CD25+regulatory T cell, Treg)在抑制脑梗死再灌注小鼠脑组织的损伤方面发挥内源性保护作用。那么,同样做为颅内缺血缺氧性损伤,调节性T细胞能否在SAH后的炎性免疫反应中发挥调节作用呢?我们利用线栓刺破法制作小鼠SAH模型,从小鼠脾、淋巴结中提取并体外扩增调节性T细胞,输入SAH小鼠体内,通过小鼠神经功能学的评价和各项实验数据的分析,探讨Treg对SAH小鼠脑保护作用的疗效。为验证体外细胞实验得到的结论,能否在动物模型中复制,并进一步探讨Treg对体外BV2及体内脑组织保护作用的机制和作用方式,我们通过脂多糖激活BV2活化的体外细胞模型,模拟体内小鼠脑组织SAH缺血缺氧的病理过程,从小鼠脾脏及淋巴结中提取并体外扩增调节性T细胞,与脂多糖激活的BV2共培养,旨在通过Treg与BV2共培养,验证Treg对体外BV2的保护作用机制。我们分别从体外细胞培养和动物在体实验两个角度,从细胞整体水平、蛋白水平和mRNA三个水平共同研究,多方位多角度综合探讨Treg发挥疗效的机制。方法：通过免疫磁珠双选的方法,提取小鼠脾脏及淋巴结Treg。颈内动脉线栓刺破法制作小鼠SAH模型,随机分为Sham组、SAH+PBS组、SAH+SP(Splenocyte,脾细胞)组、SAH+Treg组,经股静脉输注CD4+CD25+Treg,造模成功后48h,记录脑血流量及各组动物行为学指标；分别制备石蜡、冰冻切片,通过不同染色法研究脑细胞形态及阳性细胞的分布情况,验证Treg输注移植对SAH小鼠脑组织的保护作用。脂多糖诱导体外BV2激活,Treg与激活的BV2共培养,采用MTT法检测体外BV2的活性；利用硝酸还原酶法和ELISA法检测炎性因子TNF-α、IL-6、NO、IL-10的含量；采用尼罗红微球吞噬实验,验证Treg对LPS诱导BV2吞噬情况的调节作用。免疫荧光双染及免疫荧光三染法,标记体外BV2和体内SAH小鼠脑组织不同极化状态M1、M2期阳性细胞表达的情况；采用实时荧光定量PCR (RT-PCR)法分别检测体外BV2和SAH小鼠脑组织M 1、M2期标志物mRNA水平的表达量；采用Western blot法分别检测体外小胶质细胞和SAH小鼠脑组织内相关信号通道TLR4/p-NFκB、 p-P38/P-ERK1/2的蛋白表达量。通过多方位多角度的综合研究,深入探讨Treg对BV2和SAH小鼠脑组织保护作用的机制。结果：Treg静脉输注移植,SAH后小鼠死亡率下降,各项生命体征及神经功能学表现情况明显好转,神经功能学评分提高,脑组织血流量改善,小鼠脑水肿情况减轻,脑实质内微动脉及基底动脉痉挛程度减轻,血管内皮细胞受损程度减轻,小鼠脑组织神经元凋亡数量减少,损伤程度降低。Treg与LPS激活的体外BV2共培养,炎性刺激因子含量明显减少,抑制因子IL-10含量增加,同时,BV2在吞噬过程中吞掉了更多的微球,Treg对LPS激活的BV2有明显保护作用。免疫荧光染色显示,Treg输注移植,体外小胶质细胞和SAH小鼠脑组织M1期标记物表达减少,M2期标记物表达上调。RT-PCR结果显示,Treg输注移植,体外小胶质细胞和SAH小鼠脑组织M 1期标记物mRNA水平的表达减少,M2期标志物mRNA水平的表达上调。Western blot结果显示,Treg输注移植,体外BV2和SAH小鼠脑组织内炎性信号通路TLR4、p-NF-κB、p-P38、p-ERK1/勺蛋白表达量明显减少。结论：1、Treg输注移植能够减轻SAH小鼠早期死亡率,改善48h局部脑血流量,有效缓解SAH后血脑屏障的破坏程度,维护脑实质内血管形态的完整性,减轻BA痉挛程度,显著减轻小鼠皮层及海马神经元损伤的程度,减轻脑组织细胞过度凋亡的程度,有助于改善SAH后小鼠神经功能学水平,促进神经元的修复,提高SAH后小鼠生活质量。2、Treg与LPS激活的BV2共培养,细胞培养液炎性刺激因子的含量减少,抑制因子的含量增加,BV2的吞噬作用增强。3、Treg输注治疗可通过减少M1极化状态标记物mRNA水平的表达,抑制小胶质细胞M1极化；增加M2极化状态标记物mRNA水平的表达,促进小胶质细胞M2极化,从而抑制炎性细胞因子的分泌,发挥脑组织的保护功效。4、Treg可能通过抑制炎性信号通路TLR4/p-NF-κB、p-P38/p-ERK1/2的激活,减少炎性刺激因子的表达。本实验创新性地将RT-PCR法和免疫荧光染色法相结合,同时研究Treg对体外BV2和动物体内小胶质细胞不同极化状态的调节作用,通过体外细胞水平的实验与在体动物模型的实验相结合,将动物的行为学观察与细胞分子水平的机制研究相结合,多方面多角度充分地探讨了调节性T细胞对组织。的保护疗效。本实验证明,Treg对体外BV2和SAH小鼠脑组织有明显的保护作用。然而,动物模型水平得出的结论,与临床人体的实际应用仍有较大距离,细胞作为一种生物学活性物质,不能像化学药物一样稳定,其保存及运输等方面存在的问题是临床应用的重要瓶颈。另外,人体活体细胞的移植,尚存在伦理及排异等多方面的问题有待进一步研究和解决。
[Abstract]:Objective: The subarachnoid hemorrhage (SAH) is a common hemorrhagic lesion of the brain, because of its high death rate and the poor prognosis of the survivors, the quality of life of the patient is decreased. The severity of cerebral vasospasm (CVS) and early brain injury (EBI) in SAH patients was confirmed to be related to the poor prognosis. Therefore, it is necessary to develop new drugs, to control the early inflammatory immune response after SAH, to reduce the pathogenicity of EBI and CVS, and to reduce the pain of patients. It has been found that regulatory T cells (CD4 + CD25 + regulatory T cell, Treg) play an endogenous protective role in the inhibition of brain tissue damage in the brain of cerebral infarction. So, can regulatory T-cells play a regulatory role in the inflammatory immune response after SAH? In order to study the effect of Treg on the brain protection of SAH mice, we made the SAH model of the mouse by means of the line-bolt puncture method, and extracted and expanded the regulatory T-cells in vitro from the spleen and the lymph nodes of the mouse, and the regulatory T cells were amplified in vitro. The effect of Treg on the brain protection of the SAH mice was discussed by the evaluation of the neurological function and the analysis of the experimental data. In order to verify the conclusion of in vitro cell experiment, it is possible to replicate in animal model, and to further study the mechanism and function of Treg on the in vitro BV2 and in vivo brain tissue protection. We activate BV2-activated in vitro cell model by lipopolysaccharides. In order to study the pathological process of SAH ischemia and hypoxia in the mouse brain, the regulatory T cells were extracted from the spleen and lymph nodes of the mouse and the regulatory T cells were expanded in vitro, co-cultured with the BV2-activated BV2, and the mechanism of the protective effect of Treg on the in vitro BV2 was verified by co-culture of Treg and BV2. From the two angles of in vitro cell culture and in vivo experiment, we studied the mechanism of Treg's therapeutic effect from the whole level, the level of protein and the three levels of mRNA. Methods: The spleen of the mouse and the lymph node Treg were extracted by double-selection of the immunomagnetic beads. The SAH model of the mouse was made by the internal carotid artery bolt puncture method. The SAH model was randomly divided into the sham group, the SAH + PBS group, the SAH + SP (Splasmote, the spleen cell) group, the SAH + Treg group, the transfemoral vein infusion of CD4 + CD25 + Treg, the cerebral blood flow of the brain and the behavioral indexes of the animals in each group. The protective effect of Treg infusion on the brain tissue of SAH mice was verified by the study of the distribution of the morphology of the brain cells and the positive cells in different staining methods. Lipopolysaccharide induced in vitro BV2 activation, Treg was co-cultured with activated BV2, and the activity of the in vitro BV2 was detected by MTT method; the content of TNF-1, IL-6, NO, IL-10 in the inflammatory factors was detected by the nitric acid reductase method and the ELISA method; To verify the effect of Treg on the phagocytosis of BV2 induced by LPS. Immunofluorescence double staining and immunofluorescence three-staining method were used to mark the expression of M1 and M2 positive cells in different polarization states of the brain tissue of in vitro BV2 and in vivo SAH mice, and the in vitro BV2 and SAH mouse brain tissue M 1 were detected by real-time fluorescence quantitative PCR (RT-PCR). The expression of marker mRNA in M2 phase was detected by Western blot. The expression of TLR4/ p-NF-B, p-P38/ P-ERK1/2 was detected by Western blot. The mechanism of the protective effect of Treg on the brain tissue of BV2 and SAH mice was discussed through a comprehensive study of multi-angle and multi-angle. Results: After the infusion of Treg vein, the mortality of the mice after SAH decreased, the performance of various vital signs and the neurological function improved, the score of the neurological function was improved, the blood flow of the brain tissue was improved, the cerebral edema of the mice was relieved, and the degree of cerebral parenchymal micro-artery and basilar artery spasm was relieved. The degree of damage to the vascular endothelial cells was reduced, and the number of neuronal apoptosis in the brain of the mice was reduced and the degree of damage was reduced. Treg was co-cultured with LPS-activated in vitro BV2, the content of inflammatory stimulating factor was significantly reduced, and the content of IL-10 was increased. At the same time, BV2 swallowed more microspheres during the phagocytosis, and Treg had a significant protective effect on LPS-activated BV2. Immunofluorescence staining showed a decrease in the expression of the marker in the M1 phase of the brain tissue of the in vitro microglia and SAH mice, and the expression of the marker in the M2 phase was up-regulated. The results of RT-PCR showed that the expression of the mRNA level of the marker mRNA in the brain tissue of the in vitro and in vitro microglia and SAH mice was decreased, and the expression of the expression of the marker mRNA in the M2 phase was up-regulated. Western blot showed that the expression of the expression of TLR4, p-NF-B, p-P38, p-ERK1/ spoon in the brain tissue of BV2 and SAH mice in vitro was significantly reduced. Conclusion:1, Treg infusion can reduce the early death rate of SAH mice, improve the local cerebral blood flow of 48 h, effectively relieve the degree of destruction of the blood-brain barrier after SAH, maintain the integrity of the blood vessel in the parenchymal vessels, and reduce the degree of BA spasm, the degree of damage to the cortex of the mouse and the neuron of the hippocampus is obviously reduced, the degree of the over-apoptosis of the brain tissue cells is reduced, the level of the neural function of the mouse after the SAH is improved, the repair of the neuron is promoted, the quality of the life of the mouse after SAH is improved, and 2, Treg is co-cultured with the LPS-activated BV2, the content of the inflammatory stimulation factor of the cell culture solution is reduced, the content of the inhibitory factor is increased, the phagocytosis of the BV2 is enhanced, and 3, the Treg infusion therapy can inhibit the polarization of the microglia M1 by reducing the expression of the mRNA level of the M1 polarized state marker, and increase the expression of the mRNA level of the M2 polarized state marker, It is possible to reduce the expression of inflammatory stimulus by inhibiting the activation of the inflammatory signal pathway TLR4/ p-NF-SupB, p-P38/ p-ERK1/2. In this experiment, RT-PCR and immunofluorescence staining were used to study the effects of Treg on the different polarization states of microglia in vitro and in vivo. By combining the behavior of animal and the mechanism of cellular molecular level, the effect of regulatory T cell on the tissue was discussed in various aspects. And the protective effect is improved. The experimental results show that Treg has a significant protective effect on the brain tissue of BV2 and SAH mice in vitro. However, the results of animal model's level show that there is still a great distance from the actual application of the clinical human body. As a kind of biological active substance, the cell can't be as stable as the chemical medicine, and the problems in the preservation and transportation of the cell are the important bottleneck of clinical application. In addition, the transplantation of living cells of human body, there are still many problems, such as ethics and exclusion, to be further studied and solved.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743.35

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