基于小胶质细胞吞噬作用探讨“脊髓康”神经保护效应的研究

发布时间：2018-03-04 01:13

本文选题：脊髓康　切入点：小胶质细胞　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：脊髓损伤(Spinal cord injury,SCI)是一种严重致残性疾病,其引起的残疾无论对患者、对家庭或对社会都是一种最严重的灾难,且痛苦和负担往往伴随终身。目前基础及临床研究认为,SCI后神经元的继发性损伤是可能被逆转的,这为SCI治疗提供了可研究方向。小胶质细胞是驻留于中枢神经系统(Central nervous system,CNS)内的免疫细胞,其为CNS的免疫与防御系统的重要组成部分,能够保护CNS免受各种病理因子的侵害,但其有时亦会导致CNS相关病变的进一步恶化。当中风、脑外伤、SCI等神经病变发生后,小胶质细胞在对损伤局部神经元表现出破坏、修复的双重作用。一方面,小胶质细胞可做出迅速的免疫应答,定向迁移至病变部位后释放大量促炎因子启动炎性反应,过度的、反复的炎症反应及有害代谢产物对神经系统造成严重的损害,导致大量神经元的继发性坏死及凋亡,影响神经元的修复、再生;另一方面,小胶质细胞迁移至损伤部位后,其吞噬作用可通过清除凋亡神经元,减少细胞因子、神经毒性物质等释放,进而抑制过度的免疫炎性反应,协调、促进神经元网络状结构的重建及功能的恢复。故而小胶质细胞在CNS生理病理变化中有着重要意义。"脊髓康"是笔者导师临床经验方,临床实践及研究证明该方有着良好的临床疗效。基于此方,我们前期系统开展了多项"脊髓康"干预SCI的基础性研究,结果表明,"脊髓康"对SCI后免疫应答有着重要的调节作用,其能有效促进损伤神经元的修复再生。目前,中医药对小胶质细胞吞噬作用的研究较少,已有研究主要局限于部分中药能够抑制小胶质细胞活化所致的过度免疫炎性反应以降低神经二次损伤的方面,基于此,本研究欲从小胶质细胞吞噬作用角度,探讨"脊髓康"有效干预SCI的潜在机制,为SCI的临床治疗提供一定科学依据。本文第一部分采用慢病毒转染绿色荧光蛋白基因标记小胶质细胞株BV2,核染色标记死亡神经元的方法,建立了一个可动态观察BV2吞噬神经元碎片的体系,研究并证实了"脊髓康"可促进BV2对神经元碎片的吞噬作用;我们认为,中枢神经系统是一个由多种胶质细胞及神经元构成的复杂体系,SCI的发生必然不是单一系统或某一细胞群的病变,而是多因素的"整体病变",故在第一部分的研究基础上,我们提取胎鼠的部分神经组织,未经特殊筛选将其接种于体外环境中培养,初步、简易的模拟了一个多因素、立体式、整体性的混杂细胞体系,在诱导混杂细胞体系中神经元凋亡后予"脊髓康"干预,再次证实了"脊髓康"对模拟的"自然混杂体系"中小胶质细胞吞噬功能的促进作用,体现了中医药"整体"干预的理念;上述两部分实验表明,"脊髓康"能有效调控小胶质细胞的吞噬功能,那么该作用到底是否与促神经修复有相关性?基于这些问题,我们开展了第三部分实验,即验证"脊髓康"是否能通过调控小胶质细胞吞噬作用影响神经元修复再生,在此实验中,我们成功分离、提纯了原代神经元、原代小胶质细胞,建立小胶质细胞/损伤神经元共培养体系,"脊髓康"含药血清间接干预共培养体系,而后以免疫荧光双重标记法观察混培养24h后小胶质细胞对神经元碎片的吞噬作用及96h后损伤神经元突起生长情况,我们发现"脊髓康"促进小胶质细胞清理凋亡神经元碎片的同时,存活神经元的修复再生能力得到了明显的提升。综上,我们得出:中药复方"脊髓康"能够有效促进小胶质细胞吞噬神经元碎片作用,可能以此改善局部微环境,促进损伤神经元的修复再生。第一部分"脊髓康"对小胶质细胞吞噬神经元碎片的影响目的:观察中药复方"脊髓康"对小胶质细胞吞噬神经元碎片的影响。方法:制备"脊髓康"含药血清,分离、培养、鉴定原代神经元,慢病毒转染绿色荧光蛋白基因标记小胶质细胞株BV2,核染色标记神经元,荧光显微镜下观察含药血清干预后小胶质细胞对神经元碎片的吞噬作用。结果:在吞噬百分率方面,"脊髓康"中、高剂量组均高于空白组(P0.05),与脂多糖组对比差异无统计学意义(P0.05);在吞噬指数方面,"脊髓康"低、中、高剂量组均高于空白组(P0.05),中、高剂量组与脂多糖组对比差异无统计学意义(P0.05)。结论:中药复方"脊髓康"能调控BV2对神经元碎片的吞噬作用,其促进损伤神经元轴突再生的机制可能与此相关。此外,本研究为中医药调控小胶质细胞干预CNS相关疾病的研究提供了新的思路及方法。第二部分"脊髓康"对自然混杂体系中原代小胶质细胞吞噬作用的影响目的:观察中药复方"脊髓康"对自然混杂体系中原代小胶质细胞吞噬神经元碎片的影响。方法:制备"脊髓康"含药血清;分离、培养原代神经细胞,建立混杂细胞体系并予鉴定;以谷氨酸诱导混杂体系中神经元损伤,以"脊髓康"含药血清干预混杂体系;以一抗β3-tubulin、二抗Alexa 594标记神经元骨架及碎片,以一抗CD11b、二抗Alexa 488标记小胶质细胞相关膜蛋白,观察"脊髓康"干预后混杂体系中小胶质细胞对神经元碎片的吞噬作用。结果:在吞噬指数方面,"脊髓康"含药血清低、中、高剂量组均高于空白组(P0.05),中、高剂量组较LPS组差异无统计学意义(P0.05);在吞噬百分率方面,中、高剂量组均高于空白组(P0.05),中剂量组低于LPS组(P0.05)。结论:抗SCI有效方"脊髓康"可调控小胶质细胞清理神经元碎片的作用,改善局部微环境,促进损伤神经元的修复再生。第三部分"脊髓康"对共培养体系中小胶质细胞吞噬及损伤神经元再生的影响目的:观察中药复方"脊髓康"对共培养体系中小胶质细胞吞噬及损伤神经元修复再生的影响。方法:制备"脊髓康"含药血清,分离、鉴定原代海马神经元、原代小胶质细胞,谷氨酸诱导神经元损伤,含药血清预处理小胶质细胞,建立小胶质细胞/损伤神经元共培养体系,采用免疫荧光双重标记法观察混培养24h后小胶质细胞对神经元碎片的吞噬作用及96h后损伤神经元突起生长情况。结果:混培养24h后,"脊髓康"含药血清中、高剂量组在吞噬指数及吞噬百分率方面均高于空白组(P0.05),中剂量组低于LPS组(P0.05),高剂量组则与LPS组无显著差异(P0.05)。混培养96h后,"脊髓康"含药血清中、高剂量组一级突起数量多于空白组(P0.05),与LPS组比较差异无统计学意义(P0.05)。"脊髓康"中、高剂量组平均突起较空白组长(P0.05),与LPS对比,"脊髓康"中剂量组平均神经突起较短(P0.05),而高剂量组则平均突起较长(P0.05)。结论:"脊髓康"通过调控小胶质细胞吞噬功能促进损伤神经元的修复与再生,这可能与其促进凋亡物质清理后,于局部微环境建立一个无形的保护屏障有关,但其详细机制仍待进一步研究。综上所述,本研究工作的主要创新之处在于:1首次从调控小胶质细胞吞噬作用的角度对中医药干预SCI进行了研究,为"脊髓康"温肾通督抗SCI作用做出了新的科学阐释。2自行研究、建立了多种能准确、有效以及动态观察小胶质细胞吞噬神经元碎片的方法,验证了"脊髓康"对小胶质细胞吞噬的调控作用,一定程度上明确了该作用与神经修复再生的相互关系。3基于中医药干预"整体"理念,及SCI整体病变的思考,体外建立了一个多因素、整体性的混杂体系,并进行了初步的研究。
[Abstract]:Spinal cord injury (Spinal cord, injury, SCI) is a serious disabling disease, its cause of disability to the patient, the family or are one of the most serious disaster to the society, and is often accompanied by pain and burden of life. The basic and clinical research that SCI induced secondary injury of neurons is likely to be reversed, this provides the research direction for the treatment of SCI. Microglia are resident in the central nervous system (Central nervous system, CNS) in immune cells, an important part of the immune defense system and CNS, can protect CNS from various pathological factors of infringement, but its sometimes will also lead to further the deterioration of diseases related to CNS. When the stroke, brain trauma, etc. SCI neuropathy, microglia in the injury of local neurons showed destruction of the dual role of repair. On the one hand, microglial cells can make rapid free Immune response, migrate to the lesion after the release of a large number of proinflammatory cytokines that initiate the inflammatory reaction, excessive, repeated inflammation and harmful metabolites cause serious damage to the nervous system, resulting in a large number of secondary neuronal necrosis and apoptosis of neurons, repair, regeneration; on the other hand, microglial cells migrate to after the injury, the phagocytosis by clearance of apoptotic neurons, reduced cytokine release, neurotoxic substances, and then inhibit the excessive inflammatory reaction, coordination, promote reconstruction and function of neural network structure recovery. Therefore microglia plays an important role in the physiological and pathological changes in CNS Jisuikang. "Is the author of my tutor's clinical experience, clinical practice and research show that the party has a good clinical effect. Based on this, we carried out a number of pre system based" Jisuikang "intervention SCI Research results show that "Jisuikang" plays an important role in the regulation of immune response after SCI, which can effectively promote the repair and regeneration of injured neurons. At present, Chinese medicine phagocytosis less research on microglia, the existing research is mainly limited to excessive immune inflammatory reaction of some traditional Chinese medicine can inhibit the activation of microglia in order to reduce two times caused by nerve injury, based on this, this research intends to small glial cell phagocytosis angle, explore potential mechanisms of Jisuikang "effective intervention of SCI, to provide a scientific basis for clinical treatment of SCI. In the first part of this paper using lentiviral marker transfected with green fluorescent protein gene of microglial cell line BV2. Nuclear staining of dead neurons, we set up a dynamic observation of BV2 neuron debris phagocytic system, and the results confirmed the" Jisuikang "can promote BV2 on neuronal debris swallow Apoptosis; we believe that the central nervous system is a complex system composed of a variety of glial cells and neurons, the occurrence of SCI is not necessarily a single system or a group of cells of the lesion, but many factors ", so the overall lesions based on the first part of the study, we extracted nerve tissue of fetal rats without special screening, were inoculated with in vitro culture environment, preliminary, simple simulation of a multi factor, three-dimensional, hybrid cell system integrity, in the induction of neuronal apoptosis after cell hybrid system to" Jisuikang "intervention, once again confirmed the role of simulated" Jisuikang "the natural hybrid system" the phagocytic function of microglia, reflects the traditional Chinese medicine "whole" intervention idea; show that the two part of the experiment, "Jisuikang" can effectively control the phagocytic function of microglia, the role of promoting and whether There is a correlation between the nerve repair? Based on these questions, we conducted third experiments, which verify the "Jisuikang" whether through the regulation of microglial phagocytosis of neuron regeneration, in this experiment, we successfully isolated and purified primary neurons, primary microglia, establishment of microglia / total neuronal injury training system, "Jisuikang" medicated serum indirect intervention co culture system, and then by double immunofluorescent staining method to observe the mixed culture of 24h microglia on neuronal debris phagocytosis and 96h after injury of neurite growth, we find that "Jisuikang" to promote microglia clear neuronal apoptosis fragments at the same time, the ability of regeneration repair of surviving neurons has been significantly improved. In summary, we conclude that: the traditional Chinese medicine "Jisuikang" can effectively promote microglia phagocytosis of neuronal debris May, in order to improve the local micro environment, promote the repair and regeneration of injured neurons. The first part "Jisuikang" phagocytosis of neurons debris on the microglial cells Objective: To observe the effect of traditional Chinese medicine "Jisuikang" phagocytic debris on microglia neurons. Methods: the preparation of "Jisuikang" serum, isolation, culture primary identification of neurons, labeled, lentiviral transfection of green fluorescent protein gene on BV2 cell line, nuclear staining labeled neurons, observe serum stem phagocytosis of microglia on neuronal prognosis fragments under fluorescence microscope. Results: the percentage of phagocytosis, "Jisuikang" in high dose group were higher than those of control group (P0.05), there was no significant difference compared with LPS group (P0.05); the phagocytic index, "Jisuikang" low, high dose group were higher than those of control group (P0.05), and the high dose group and LPS group contrast There was no statistically significant difference (P0.05). Conclusion: the traditional Chinese medicine "Jisuikang" phagocytosis regulation of BV2 on neuronal debris, the damage mechanism of promoting axonal regeneration may be related to this. In addition, provides new ideas and methods of this research for the regulation of Chinese medicine intervention of microglia CNS related diseases the second part "Jisuikang. Effect of natural hybrid system primary microglial phagocytosis Objective: To observe the effect of traditional Chinese medicine" Jisuikang "of natural hybrid system primary microglial phagocytosis of neuronal debris. Methods: the preparation of" Jisuikang "containing serum; separation of primary cultured nerve cells, hybrid and to establish the system of cell identification; neuronal damage induced by glutamate in the hybrid system, hybrid system to intervention" Jisuikang "to a drug containing serum; anti beta 3-tubulin, two anti Alexa 594 labeled neurons and fragments of skeleton In two, anti CD11b, anti Alexa 488 labeled microglia associated membrane protein, observe the "Jisuikang" stem phagocytosis prognosis hybrid system of microglia on neuronal debris. Results: the phagocytic index, "Jisuikang" serum containing low and high dose group were higher than those of control group (P0.05) in the high dose group, the difference was not statistically significant compared with LPS group (P0.05); the phagocytic percentage, in high dose group were higher than those of control group (P0.05), middle dose group was lower than that of LPS group (P0.05). Conclusion: Anti SCI effective "Jisuikang" regulation of microglia neuron debris cleanup the role, improve the local micro environment, promote the repair and regeneration of injured neurons. In the third part, "Jisuikang" on the effect of co culture system of microglial phagocytosis and injury neuron regeneration Objective: To observe the traditional Chinese medicine "Jisuikang" system of microglial phagocytosis and damage God co culture The influence of element repair and regeneration. Methods: the preparation of "Jisuikang" serum, separation, identification of primary hippocampal neurons, primary microglia neuron injury induced by glutamate, serum pretreatment of microglia, establishment of microglia / damaged neurons co culture system using double immunofluorescent staining method to observe the mixed after 24h cultured microglia on neuronal phagocytosis and 96h fragmentation after injury neuronal growth. Results: the mixed culture of 24h, "Jisuikang" medicated serum in high dose group in the phagocytic index and phagocytic percentage were significantly higher than those in control group (P0.05), middle dose group was lower than that of LPS group (P0.05), the high dose group had no significant difference with LPS group (P0.05). The mixed culture of 96h, "Jisuikang" medicated serum in high dose group level raised more than the number of blank group (P0.05), compared with the LPS group showed no significant difference (P0.05) "Jisuikang" In the high dose group compared with blank group average projection (P0.05), compared with LPS, "Jisuikang" middle dose group the average neurite shorter (P0.05), and high dose group (P0.05) the average long protuberances. Conclusion: the "Jisuikang" through the regulation of microglial phagocytosis promoting repair and regeneration. The damage of neurons, which may be related to apoptosis of material after cleaning, in the microenvironment of the establishment of an invisible protective barrier, but the detailed mechanism still need further research. To sum up, the main innovation of this research lies in: 1 for the first time from the regulation of microglial phagocytosis of SCI was studied in the intervention of traditional Chinese medicine, made a new scientific explanation to.2 research as "Jisuikang" and warming kidney and activating anti SCI effect, creating a variety of methods can accurately, effectively and dynamically observe the microglial phagocytosis of neuronal debris, verify the "Jisuikang" on small Regulation of phagocytosis of glial cells, to a certain extent the effect of nerve regeneration and repair the relationship between the.3 of Chinese medicine intervention "based on the overall concept, thinking and the whole SCI lesions in vitro, the establishment of a multi factor, hybrid system integrity, and conducted a preliminary study.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

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