A151抑制缺氧BMDM的炎症及其在脑缺血性损害中的治疗潜能

发布时间：2018-05-08 02:21

本文选题：寡核苷酸 + 炎性小体　；参考：《山东大学》2016年博士论文

【摘要】：研究背景脑卒中是目前对人类危害最严重的疾病之一。脑卒中是高发病率、高致残率、高复发率、高死亡率、高经济负担的“五高”疾病。在我国,脑卒中是位列第一的死亡原因。免疫炎症在卒中的发生发展的病理发展过程中起重要作用。炎症是机体对刺激做出的一种防御反应,在生物体内起重要作用。当受到刺激因子的刺激时,会诱发炎症反应,从而修复组织,抵抗损伤。炎症也会对机体产生不良影响,导致免疫系统紊乱。已经知道炎症和免疫与卒中尤其是缺血性脑卒中的多种危险因素相关,包括高血压、动脉粥样硬化、糖尿病、房颤、吸烟所致的血管损害等等。更为有趣的是脑血管病的事件导致了损伤相关的分子模式(damage associated molecular patterns, DAMPs)的释放,从而增强了先天免疫反应,最后导致脑损害和有关的神经功能的缺失。目前公认的急性缺血性脑卒中的有效的治疗方法是在有效的时间窗内尽早恢复脑灌注,如临床上应用重组组织型纤溶酶原激活剂(recombinant tissue plasminogen activator, rTPA)进行超早期静脉溶栓,临床效果好,但是却受治疗时间窗、病人的一般情况及溶栓后容易并发出血等的各种并发症的多种因素的制约,所以只有大约不到20%的病人能获益,尽早恢复脑灌注在临床的实际治疗中存在困难。寻求有效的脑保护治疗成为多年研究的重点。几十年来大量的研究者专注于的细胞保护作用机制的研究,使脑细胞在缺血缺氧应激后仍能保持细胞内稳态的动态平衡,从而保护脑细胞不被破坏。实验在动物体内均获得很好的成效,但在临床转化研究中却均失败。尽管如此,在国际卒中组织的努力下,卒中的病理发展过程方面的研究仍有很大的进步。炎性小体(Inflammasomes)是一种多蛋白复合体,作为先天免疫的一部分可以在应激和感染等感受到外界信号刺激的情况下激活半胱天冬氨酸蛋白水解酶-1(caspase-1),从而产生白介素1β(IL-1β)和白介素18(IL-18)。Caspase-1和IL-1β增强炎症反应和细胞死亡,IL-1β在很多疾病包括脑缺血性损害过程中起重要作用,其机制尚未揭示。在卒中实验中,随着脑缺血的发生,IL-1β的表达增多,而且多个实验证实,阻止IL-1β的产生具有脑保护作用。值得注意的是人类的缺血性脑卒中血液和脑脊液中均可发现IL-1β的水平增高。在鼠模型中,脑缺血性损害后炎性小体相关蛋白水平增高,降低炎性小体相关蛋白水平可以减轻损害的程度。受损的细胞,包括神经细胞可以释放出三磷酸腺苷(ATP)、尿苷三磷酸(UTP),激活小胶质细胞或巨噬细胞上的离子通道P2X7受体,从而导致炎症因子前体的产生。Caspasel嵌合在大分子蛋-NLRP3上,在Caspasel的作用下,成熟的IL-1β、IL-18由其前体转化而来。缺血损害以及细胞的死亡导致了DAMPs的形成。DAMPs包括高迁移率族蛋白1(HMGB1)、热休克蛋白HSP60、A β,可以作用于细胞表面的Tool样受体(TLR),从而使炎性前体的基因表达增多。细胞因子的产生及补体的形成引起了白细胞浸润的增加,扩大了脑的损害,这样能产生更多的DAMPs,进一步加重损害。同时由损害细胞释放的抗原也激活T细胞介导的适应性的免疫。这些近期的发展使得需要再评估炎症和免疫在卒中病理生理过程中的作用。2009年诺贝尔奖的获得者发现了染色体末端的端粒帽端粒帽能保护染色体防止其融合、降解和重组等,为衰老和癌症的研究奠定了基础。哺乳动物的端粒由TTAGGG碱基对组成,这些碱基对从死亡的宿主细胞中释放并且能减轻引起组织损害的炎症反应。合成的寡核苷酸A151是由四个TTAGGG碱基对结合在硫代磷酸酯骨架上构成。A151复制了端粒DNA调节炎症反应的能力,包括调节白介素6(IL-6)、白介素12(IL-12)、γ干扰素(IFNγ)、巨噬细胞炎症蛋白2(MIP-2)和肿瘤坏死因子α(TNFα)的产生。A151作为一种的抗炎症反应制剂,其治疗潜能已经在关节炎、内毒素休克、刀豆蛋白A诱导的暴发性肝炎、眼部炎症、狼疮肾炎、动脉粥样硬化和二氧化硅所致的肺部炎症等动物模型中得到证实。至关重要的是,硫代磷酸寡核苷酸的的药效学和安全性已经在多个临床试验中得到证实。A151对脑缺血后的炎症免疫反应的影响以及对脑缺血是否具有保护作用目前尚无人研究,基于IL-1β在脑缺血性损害中的作用以及A151成功改进多种疾病炎性因子的基础上,如果A151能减轻脑缺血后的炎症免疫反应,并对脑缺血损害具有保护作用,将为缺血性脑卒中的治疗提供新的亮点。脑缺血损害时,骨髓巨噬细胞和小胶质细胞是IL-1β的主要来源。脑缺血后,除了血管周围的巨噬细胞,单核细胞迅速渗入大脑也成为巨噬细胞。所以本实验选用骨髓巨噬细胞在细菌脂多糖(LPS)和糖氧剥夺(OGD)双重处理来模拟脑内缺血缺氧状态。易卒中自发性高血压大鼠(SHR-SP大鼠)生存期短,易发卒中,是研究脑卒中最为理想的动物模型。本实验采用SHR-SP大鼠的永久性大脑中动脉离断手术模型进行研究。本实验所应用的A151及对照剂寡核苷酸C151(5'-TTCAAATTCAAATTCAAATTCAAA-3')由美国食品药物管理局(FDA)生物制品评价和研究中心(CBER)提供。目的1.体外部分：将骨髓巨噬细胞予以LPS和OGD的双重协同处理,以求模拟脑内的缺血缺氧的环境,检测A151在缺氧缺糖损害后的骨髓巨噬细胞的免疫炎症反应中的作用,能否减少炎性因子的释放,减轻炎症反应从而并减轻细胞的损害。2.体内部分：应用SHR-SP大鼠的永久性大脑中动脉离断手术模型,将从动物的行为、组织、免疫、基因等水平分别来观察A151对发生卒中后的SHR-SP大鼠的炎性因子的影响以及对脑组织的保护作用,观察其治疗潜能,以求进一步的脑保护作用。方法1.骨髓巨噬细胞的培养,取SHR-SP大鼠的股骨胫骨,冲洗骨髓腔,将得到的细胞悬液进行培养,并应用巨噬细胞特异性蛋白抗体染色,在荧光显微镜下确定99%以上的细胞都是BMDM细胞。2.应用A151、C151、LPS预处理骨髓巨噬细胞后,行OGD实验,18小时后收取上清及细胞留用。一部分上清用于做酶联免疫吸附测定((enzyme-linkedimmunosorbent assay, Elisa),一部分上清浓缩后测量蛋白浓度,和收集的裂解的细胞蛋白一起进行免疫印记实验(Western blot)。3.应用A151、C151、生理盐水分别预处理SHR-SP大鼠,对大鼠行永久性大脑中动脉离断手术,术后48小时进行神经功能评分,处死大鼠,收集脑、脾、肾血液等组织留用。4.收集的一部分大脑切片染色,应用Image J (NIH, Bethesda, MD)软件测量大鼠的脑梗死体积。5.一部分脑组织提取RNA,做Real-time PCR,检测脑中NLRP3、Aim2、NLRP4的mRNA的表达。6.收集的血液进行抗凝处理,一部分进行血液化学物质检测以及血细胞计数,另一部分离心后收集血浆进行酶联免疫检测。7.收集的一部分脑及脾组织裂解后测量蛋白浓度,做Western blot以及Elisa。8.应用JC-1试剂盒观察线粒体膜电位。结果1.A151能够明显减少细胞培养液上清中的IL-1β、IL-1 a、IL-6, CINC1和TNFY的水平,和对照组的ODN C151相比,C151只能减少IL-6、CINC1的水平,对其他因子没有影响。A151能够明显提高缺血缺氧状态下BMDM的存活率。这一结果也证实了其他研究者提出的A151在炎症细胞因子的中的作用并没有细胞毒性作用的说法。2.通过蛋白免疫印迹法来进一步检测A151在IL-1βB的成熟和表达中的作用。结果发现A151可以减少用OGD处理后的BMDM的培养液上清中的成熟的IL-1β的表达。而且A151减少了成熟的·aspasel和NLRP3以及iNOS的表达,但是A151对ASC, AIM2, NLRP1和NLRC4没有影响。3.线粒体功能障碍与NLRP3炎性小体的激活有关,本实验通过JC一1试剂盒检测发现A151能减轻线粒体膜的去极化从而减少细胞的死亡。4.在实验中应用永久性大脑中动脉离断手术模型研究A151的治疗潜能作用发现,与C151组和生理盐水组相比较,术前三天,术前一天以及术后三小时腹腔注射A151组的脑梗死体积均显著缩小,具有统计学意义。5.提取术后48小时后SHR-SP大鼠的脑组织的RNA,做Real-time PCR发现A151处理后的NLRP3的mRNA的表达明显减少,而Aim2和NLRP4的mRNA的表达无明显变化。结论1.A151减少LPS和OGD处理后的BMDM的死亡及炎性因子的释放。2.A151可以减少OGD和LPS的处理下的BMDM内的成熟的IL-1β和caspasel,并且能够减少NLRP3 and iNOS的表达。3.A151能减轻BMDM细胞的线粒体膜的去极化。4.A151可以减轻永久性大脑中动脉离断手术的SHR-SP大鼠的脑的缺血性损害。5.A151能减少永久性大脑中动脉离断手术的SHR-SP大鼠的脑内的NLRP3的mRNA的表达。
[Abstract]:Stroke is one of the most serious diseases for human being. Stroke is a high incidence, high rate of disability, high recurrence rate, high death rate, and high economic burden of "five high" disease. In China, stroke is the first cause of death. Immune inflammation plays an important role in the pathological process of the development of stroke. Inflammation is an organism's defense response to stimulation, which plays an important role in the organism. When stimulated by an irritation factor, it induces an inflammatory response to repair tissue and resist injury. Inflammation also causes adverse effects on the body and leads to immune system disorders. A variety of risk factors, including hypertension, atherosclerosis, diabetes, atrial fibrillation, vascular damage caused by smoking, and so on. More interestingly, cerebrovascular events lead to the release of damage associated molecular patterns (DAMPs), which enhances the innate immune response and eventually leads to brain damage. The effective treatment of acute ischemic stroke is to restore cerebral perfusion as early as possible in an effective time window, such as the clinical application of recombinant tissue type plasminogen activator (recombinant tissue plasminogen activator, rTPA) for ultra early venous thrombolysis, but the clinical effect is good, but the clinical effect is good, but the clinical effect is good, but the clinical effect is good, However, it is restricted by many factors such as the time window of treatment, the general situation of the patients and the complications such as bleeding after thrombolytic, so only about less than 20% of the patients can benefit. It is difficult to recover cerebral perfusion as early as possible in the clinical practice. The researchers focused on the study of the mechanism of cell protection, which kept the dynamic balance of the homeostasis in the cells after the ischemic and anoxic stress, thus protecting the brain cells from being destroyed. The experiment was successful in the animal body, but failed in the clinical transformation study. Nevertheless, in the International Stroke Organization Efforts have made great progress in the study of the pathological process of stroke. The inflammatory small body (Inflammasomes) is a polyprotein complex. As a part of the innate immunity, the cystine aspartate protein hydrolase -1 (caspase-1) can be activated by stress and infection to stimulate the external signal, thus producing interleukin 1 beta (IL-1 beta) and interleukin 18 (IL-18).Caspase-1 and IL-1 beta enhance inflammatory response and cell death. IL-1 beta plays an important role in many diseases, including cerebral ischemic damage. The mechanism has not been revealed. In the stroke experiment, the expression of IL-1 beta is increased with the occurrence of cerebral ischemia, and many experiments have confirmed that the production of IL-1 beta has the brain. It is worth noting that the levels of IL-1 beta in the blood and cerebrospinal fluid of human ischemic stroke are elevated. In the rat model, the levels of inflammatory small body related proteins after ischemic brain damage increase and the levels of inflammatory small body related proteins reduce the degree of damage. The damaged cells, including the nerve cells, can be released. Adenosine triphosphate (ATP), uridine three phosphoric acid (UTP), activates the ion channel P2X7 receptor on the microglia or macrophages, leading to the production of.Caspasel in the precursor of the inflammatory factor on the large molecular egg -NLRP3. Under the action of Caspasel, the mature IL-1 beta, IL-18 is converted from its precursors. Ischemia damage and cell death are caused by the action of Caspasel. The formation of DAMPs.DAMPs includes high mobility group protein 1 (HMGB1), heat shock protein HSP60, A beta, which can act on the Tool like receptor (TLR) on the surface of the cell, thereby increasing the gene expression of the inflammatory precursors. The production of cytokines and the formation of complement cause the increase of leukocyte infiltration and the damage of the brain, which can produce more DAM. Ps, further aggravating the damage. At the same time, the antigen released by the damaged cells also activates the adaptive immunity mediated by T cells. These recent developments have led to the need to reassess the role of inflammation and immunity in the pathophysiological process of stroke. The Nobel prize winner of the Nobel prize found the telomere hat at the end of the chromosome to protect the chromosome. To prevent its fusion, degradation and recombination, it lays the foundation for the study of aging and cancer. The mammalian telomere is composed of TTAGGG base pairs, which are released from the dead host cells and can reduce the inflammatory response to tissue damage. The synthesized oligonucleotide A151 is combined with four TTAGGG bases in thiosphosphates. On the skeleton,.A151 replicates the ability of telomere DNA to regulate the inflammatory response, including the regulation of IL-6 (IL-6), interleukin 12 (IL-12), interferon gamma (IFN gamma), macrophage inflammatory protein 2 (MIP-2) and tumor necrosis factor alpha (TNF alpha) as an anti inflammatory reaction agent, and its therapeutic potential has been in arthritis, endotoxic shock, and knife A induced viral hepatitis, ocular inflammation, lupus nephritis, atherosclerosis, and silica induced pulmonary inflammation are confirmed. It is essential that the pharmacodynamics and safety of thiophosphoric acid oligodeoxynucleotides have been confirmed in multiple clinical trials to confirm the inflammatory response of.A151 to cerebral ischemia On the basis of the role of IL-1 beta in ischemic brain damage and the success of A151 to improve the inflammatory factors of various diseases, if A151 can reduce the inflammatory response after cerebral ischemia and protect the cerebral ischemic damage, it will be ischemic stroke. Treatment provides a new bright spot. Bone marrow macrophages and microglia are the main sources of IL-1 beta during cerebral ischemia. After cerebral ischemia, monocytes are rapidly infiltrated into the brain and macrophages except the macrophages around the blood vessels. Therefore, bone marrow macrophages are used in the double treatment of LPS and OGD. To simulate the state of cerebral ischemia and hypoxia. The survival period of the stroke prone spontaneously hypertensive rats (SHR-SP rats) is short and prone to stroke. It is the most ideal animal model for the study of stroke. This experiment used the permanent middle cerebral artery disconnection model in SHR-SP rats. The A151 and the control agent oligonucleotide C151 (5'-TTCA AATTCAAATTCAAATTCAAA-3') is provided by the American food and Drug Administration (FDA) biological products evaluation and Research Center (CBER). Objective 1. in vitro part: Double synergistic treatment of bone marrow macrophages with LPS and OGD in order to simulate the ischemic and anoxic environment in the brain and to detect the immune inflammation of bone marrow macrophages of A151 after hypoxia and glucose deficiency. The role of the reaction to reduce the release of inflammatory factors, alleviate the inflammatory response and reduce the cell damage in.2. body parts: the permanent middle cerebral artery dissection operation model of SHR-SP rats will be used to observe the inflammatory causes of the SHR-SP rats after apoplexy, respectively, from the animal behavior, tissue, immunity, and gene levels. The effect of the child and the protective effect on the brain tissue, observe its therapeutic potential in order to further protect the brain. Method 1. bone marrow macrophages of 1. bone marrow were cultured, the femur tibia of the rats was taken and the bone marrow cavity was flushed. The cell suspension was cultured, and the macrophage specific protein antibody staining was used to determine 9 under the fluorescence microscope. More than 9% of the cells were BMDM cells.2. using A151, C151, LPS pretreatment of bone marrow macrophages, OGD experiment, 18 hours after the collection of supernatant and cell retention. Part of the supernatant for enzyme linked immunosorbent assay (enzyme-linkedimmunosorbent assay, Elisa), a division of the concentration of protein concentration after concentration, and the collection of lysed cells The immune imprinting experiment (Western blot).3. used A151, C151, and physiological saline to pretreat SHR-SP rats respectively. The rats were treated with permanent middle cerebral artery disconnection, and the nerve function score was performed 48 hours after the operation. The rats were killed and some tissues of brain, spleen, kidney and blood were collected for.4. collection and a part of the brain was stained and Image was applied. J (NIH, Bethesda, MD) software to measure the volume of cerebral infarction in rats.5. a part of the brain tissue extracted RNA, Real-time PCR, the expression of NLRP3, Aim2, NLRP4 mRNA in the brain for anticoagulant treatment, a part of blood chemical substances and blood cell count, and the other part of centrifugation to collect plasma for enzyme linked immunization The concentrations of protein in the brain and spleen tissues collected by.7. were measured, and the mitochondrial membrane potential was observed by Western blot and Elisa.8. using JC-1 kit. Results 1.A151 could significantly reduce the level of IL-1 beta, IL-1 a, IL-6, CINC1 and TNFY in the supernatant of cell culture. Level, no effect on other factors,.A151 can significantly increase the survival rate of BMDM under ischemic and anoxic state. This result also confirms the role of other researchers in the role of A151 in inflammatory cytokines and does not have cytotoxic effects..2. further detects the maturity and table of A151 in IL-1 beta B through protein immunoblotting. The results showed that A151 could reduce the expression of mature IL-1 beta in the supernatant of BMDM culture after OGD treatment. Moreover, A151 reduced the expression of mature, aspasel, NLRP3 and iNOS, but A151 has no effect on the activation of ASC, AIM2, NLRP1, and iNOS. This experiment is related to the activation of the inflammatory small body. JC 1 Kit detection showed that A151 could reduce the depolarization of mitochondrial membrane and reduce cell death..4. was used to study the therapeutic potential of A151 with permanent middle cerebral artery disconnection model, and compared with C151 group and saline group, three days before operation, one day before operation and three hours after operation to intraperitoneal injection of A151. The volume of cerebral infarction in the group was significantly reduced, with statistical significance of RNA in the brain tissue of SHR-SP rats 48 hours after.5. extraction. Real-time PCR found that the expression of mRNA in NLRP3 after A151 treatment was significantly reduced, but the expression of Aim2 and NLRP4 mRNA was not significantly changed. The release of.2.A151 can reduce the mature IL-1 beta and caspasel within the BMDM of the BMDM and reduce the NLRP3 and iNOS expression.3.A151 can reduce the mitochondrial membrane depolarization.4.A151 of the BMDM cells to reduce the ischemic damage to the brain of the permanent middle cerebral artery dissection operation. The expression of NLRP3 mRNA in the brain of SHR-SP rats after surgery for persistent middle cerebral artery occlusion.

【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】：R743.3

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