血清因子白蛋白和补体在中枢神经系统损伤后炎症反应中的作用研究

发布时间：2018-01-05 04:17

本文关键词：血清因子白蛋白和补体在中枢神经系统损伤后炎症反应中的作用研究　出处：《第三军医大学》2009年博士论文　论文类型：学位论文

【摘要】： 目的:在包含创伤在内的中枢神经系统(Central nervous system, CNS)病变中,血脑屏障(Blood-brain barrier, BBB)的开放会引起多种血液成分进入CNS,进而可能在CNS引起相应的效应。而炎症反应是中枢系统微环境发生改变后的重要病理改变。研究发现BBB受损区域多伴有持续活化的炎症反应存在。而持续过度的炎症反应在许多中枢病变中都造成了损害性的后果。因此,探究BBB损伤性CNS病变中炎症反应持续激活的机制,设法对其进行调控,有可能达到降低损害,改善愈后的目的。血清白蛋白是血清中含量最丰富的蛋白质,约占血清蛋白质总量的50%。但在CNS却近乎缺失。BBB损伤后大量的血清白蛋白进入CNS已被证实,然而其在中枢神经系统引起的效应却鲜有报道。因此,本实验将首次研究血清白蛋白作用于中枢神经系统主要的炎症效应细胞——小胶质细胞后对其活化增殖和分泌促炎症细胞因子的影响,明确其对中枢炎症反应的激活作用。补体系统是存在于人和脊椎动物血清与组织液中一组具有酶活性的蛋白,广泛参与机体的防御反应和免疫调节,也可介导免疫病理如损伤性炎症反应,是体内具有重要生物学意义的效应系统和效应放大系统。正常CNS中补体表达水平极低。研究发现血清中的补体成分在CNS损伤后穿过受损的BBB会介导和加重CNS的炎症反应;而炎症反应又是造成CNS损伤后继发性损伤和胶质瘢痕形成的重要机制。鉴于C3是补体活化途径中的中心因子,我们拟利用C3基因敲除小鼠的脊髓损伤模型,观察抑制补体活化能否减轻SCI小鼠脊髓的继发性损伤,促进功能恢复,并初步探讨其机制。方法: 第一部分:小胶质细胞是存在于CNS的天然免疫细胞,当CNS微环境发生变化时,小胶质细胞可迅速活化增殖;而促炎症细胞因子IL-1β和TNF-α是介导CNS炎症反应的重要炎症介质。因此,我们以0.5 mg/ml的血清白蛋白刺激N9小胶质细胞株,设PBS对照组,通过RT-PCR和Real-time RT-PCR方法检测干预后3、6、9、12h小胶质细胞IL-1β和TNF-αmRNA表达情况;分别以0、0.1、0.5、1.0、2.0 mg/ml血清白蛋白和LPS刺激N9小胶质细胞,于24h收集各组细胞上清进行ELISA检测,测定IL-1β和TNF-α细胞因子的浓度,了解其对小胶质细胞分泌IL-1β和TNF-α作用的量效关系;以0.5 mg/ml的血清白蛋白稀释液刺激N9小胶质细胞株,设PBS对照组,分别于3、6、12、24h收集血清白蛋白刺激组和PBS对照组细胞上清进行ELISA检测,测定IL-1β和TNF-α细胞因子的浓度,了解其作用的时间特点;将0.5μl 100 mg/ml血清白蛋白注入C57BL/6J小鼠运动皮层,采用免疫组化法在体观察白蛋白作用后小胶质细胞的活化程度。第二部分:为准确判断干预手段对脊髓损伤后功能结局的影响,我们需要稳定、重复性好、易操作的动物模型作为研究平台。由于目前的基因修饰动物以小鼠为主,因此我们以C57BL/6J小鼠为实验动物,建立脊髓夹伤模型,并对其特点进行评价,为下一步的研究奠定基础。我们采用BMS后肢运动评分观察夹伤致0.1、0.25、0.5 mm的不同分级损伤程度对小鼠后肢功能损伤及恢复的影响;并以HE染色观察不同损伤程度致脊髓损伤后8w时脊髓的病理表现;以HE染色观察SCI后小鼠脊髓3d、1w、2w、4w时脊髓的病理改变特点;以GFAP免疫组织化学染色观察SCI后3d、1w、2w、4w、8w时小鼠脊髓星形胶质细胞反应特点;以BDA顺行示踪观察小鼠SCI后8w神经纤维再生特点及其与胶质瘢痕的关系;以流式细胞计数法检测SCI后3d、1w、2w、8w的小胶质细胞/巨噬细胞的活化特点。第三部分:采用上述建立的小鼠脊髓夹伤模型,以C3-/-小鼠及其野生对照C3+/+小鼠为实验动物,通过BMS评分评估后肢运动功能、HE染色观察脊髓病理改变、流式细胞术检测小胶质细胞/巨噬细胞活化、免疫组化和Western blot技术检测星形胶质细胞GFAP表达,观察对比C3基因敲除对小鼠SCI后功能恢复的影响并初步探讨其机制。结果: 第一部分: 1.与PBS对照组细胞相比,0.5 mg/ml的血清白蛋白刺激的小胶质细胞3、6、9、12h的IL-1β和TNF-αmRNA表达均显著增加; 2.0.1、0.5、1.0、2.0 mg/ml白蛋白刺激24h后小胶质细胞培养上清中IL-1β、TNF-α的浓度随血清白蛋白浓度的增加而增加,2.0 mg/ml刺激组IL-1β、TNF-α浓度最高;各白蛋白刺激组IL-1β浓度和TNF-α浓度均显著高于0 mg/ml组;LPS刺激组亦显著高于未刺激组。 3.0.5 mg/ml的血清白蛋白作用后,小胶质细胞分泌IL-1β随时间增加而增多,且于6h后各时间点均显著高于对照组;小胶质细胞分泌TNF-α随时间增加而增多,且于3h开始及其后时间点均显著高于对照组。 4.小鼠皮层注射血清白蛋白引起小胶质细胞活化的范围和数量明显强于PBS对照组。第二部分: 1.成功建立了稳定的C57BL/6J小鼠脊髓夹伤动物模型,BMS评分检测显示SCI后各组小鼠后肢运动功能有不同程度损伤,之后持续8w的行为学观察中虽都有恢复,但不同致伤程度组小鼠恢复情况有显著性差异。夹伤越轻(至0.5mm),恢复越好;夹伤越重(至0.1mm),恢复越差。 2.不同致伤程度小鼠8w时的脊髓病理改变与行为学结果相符。夹至0.1mm组小鼠的脊髓损伤范围最广,可见夹伤处大量炎细胞浸润形成的瘢痕及微小的空洞形成;随着夹伤程度的减弱,瘢痕的范围逐渐减小,残留的正常组织逐渐增多。 3.损伤后4w的连续病理观察发现:伤后3d,脊髓以变性、坏死为主要表现;1w时已有大量炎细胞浸润,并且出现继发性损伤区域,可见损伤两侧水肿形成较多空泡;2w时仍有大量炎细胞浸润,继发损伤和水肿区域较1w时缩小;4w时仅残留炎细胞浸润,继发损伤表现明显减弱,组织进入修复期。 4.损伤后8w的星形胶质细胞连续观察发现:伤后3d,星形胶质细胞尚未有明显的活化表现;1w时星形胶质细胞肥大,突起增粗增长,呈明显的活化状态,范围延伸至损伤区的头尾两侧;2w时星形胶质细胞的数量较1w时有所增多,但是细胞及突起的形态已不如7d时肥大明显;4w和8w时星形胶质细胞的活化程度和范围明显减弱,但在损伤中间处瘢痕组织的周边仍包绕有较多形态肥大,突起增多的反应型星形胶质细胞形成胶质瘢痕,而稍远处的星形胶质细胞形态已基本接近正常。 5.BDA顺行示踪发现SCI后10w+2d,夹伤至0.25 mm组小鼠脊髓中,大部分BDA标记的皮质脊髓束到达夹伤处的头侧端,但是无法继续下行,在夹伤处头侧端形成多个圆形的终球,仅有少数BDA着色的神经纤维在原皮质脊髓束位置的背侧方通过夹伤处,到达夹伤处尾侧端。经GFAP复染后,可见BDA终球形成位置恰为星形胶质细胞肥大增生形成胶质瘢痕处。 6.流式细胞术检测CD68阳性细胞提示损伤处小胶质/巨噬细胞于伤后3d即明显活化增殖且3d时活化状态已很强,其数量在7d达到高峰后,一直持续到伤后8w均存在活化状态的小胶质/巨噬细胞。第三部分: 1.BMS评分显示C3-/-小鼠夹伤后的后肢功能恢复明显好于C3+/+组小鼠。 2.HE染色提示夹伤后4w,C3-/-小鼠脊髓损伤处的炎症细胞浸润形成瘢痕的范围明显少于C3+/+组小鼠,而残留的正常组织却要多于C3+/+小鼠。 3.流式细胞检测CD68阳性细胞提示伤后3d、7d,C3-/-小鼠损伤处的小胶质/巨噬细胞活化程度明显弱于C3+/+组小鼠。 4.免疫组化和Western blot检测均提示C3-/-小鼠伤后4w GFAP的表达明显弱于C3+/+组小鼠。结论: 1.本研究证实血清白蛋白能刺激小胶质细胞的活化增殖,并能促进其分泌IL-1β、TNF-α促炎症细胞因子,提示血清白蛋白在CNS炎症反应中可能起重要的活化作用。 2.本研究建立了稳定、易操作、重复性好的小鼠SCI模型,既适用于SCI急性期小胶质细胞活化炎症反应的研究,也可用于SCI慢性期星形胶质细胞形成胶质瘢痕及神经再生的研究。 3.补体系统可能通过加重炎症反应,并促进星形胶质细胞瘢痕的形成,对小鼠脊髓损伤的修复产生不利影响。综上所述,本文通过对血清白蛋白和补体的研究,进一步丰富了血清因子对中枢神经系统损伤后炎症反应作用的认识,并为通过干预炎症反应治疗中枢神经系统病变提供了理论依据。
[Abstract]:Objective: the central nervous system in the wound, (Central nervous system, CNS) lesions, blood brain barrier (Blood-brain barrier BBB) open will cause various blood components into the CNS, which may cause the corresponding effect in CNS. Inflammation is an important pathological change of central nervous system micro environment changes. The study found that the presence of inflammation associated with impaired BBB regional persistent activation of inflammation. Excessive and persistent in many central lesions have caused damage of consequences. Therefore, the research of mechanism of BBB damage of CNS lesions in the inflammatory response to sustained activation, trying to regulate its, is likely to reduce the damage, to improve after healing.
Serum albumin is the most abundant protein in serum, total serum protein 50%. in CNS is a near absence of.BBB damage of serum albumin into a CNS has been confirmed, but the effect caused in the central nervous system has been rarely reported. Therefore, this experiment will be the first study of serum albumin in inflammatory effector cells the main central nervous system: after the activation of microglia proliferation and secretion of proinflammatory cytokines and the inflammatory reaction of central nervous system activation.
The complement system is present in human serum and tissue fluid in vertebrates and a group of proteins with enzymatic activity, immune defense reaction and widely involved in regulation, but also induce immune pathological injury such as inflammatory reaction, is the effect of system and has important biological significance, the effect should be amplified system. Complement expression in normal CNS level very low. The study found that the complement components in serum through BBB damage after CNS damage will be mediated by inflammation and exacerbation of CNS; inflammation can cause secondary injury and glial scar formation after injury. CNS in C3 is the complement activation factor pathway in our center, quasi knockout the mouse model of spinal cord injury by C3 gene, the secondary injury can reduce the SCI activation inhibition was observed in the mouse spinal cord complement, promote functional recovery, and to explore its mechanism.
Method:
The first part: microglial cells are innate immune cells existed in CNS, when the CNS micro environment changes, microglia can be activated and proliferation; proinflammatory cytokines IL-1 and TNF- alpha beta is an important medium of inflammation mediated inflammatory reaction in CNS. Therefore, we use serum albumin stimulation of 0.5 mg/ml N9 glial cells, the control group with PBS, RT-PCR and Real-time by RT-PCR assay after intervention 3,6,9,12h microglia IL-1 beta and TNF- alpha mRNA expression respectively; the serum albumin and LPS 0,0.1,0.5,1.0,2.0 mg/ml stimulated N9 microglia in 24h culture supernatant was collected for detection of ELISA, IL-1 and TNF- concentration determination of beta alpha cell factor the understanding of the dose-response relationship between the secretion of IL-1 and TNF- alpha beta effects on microglia; the serum albumin of 0.5 mg/ml dilution stimulation of N9 cells, the control group with PBS, respectively, 3,6,12,2 4h serum albumin stimulation group and PBS control group cells were detected by ELISA, IL-1 and TNF- concentration determination of beta alpha cytokines, understand the characteristics of the time effect; 0.5 l 100 mg/ml serum albumin injection in mice C57BL/6J cortex, activation of microglia was detected by immunohistochemical method and albumin in vivo after.
The second part: to accurately determine the effect of interventions on functional outcome after spinal cord injury, we need stability, good reproducibility and easy operation of the animal model as the research platform. Because of a genetically modified animal at present by mice, so we used C57BL/6J mice as experimental animals, a spinal cord injury model, and to evaluate the the characteristics, lay the foundation for the next research. The degree of injury of different grades by BMS hind limb crush induced by 0.1,0.25,0.5 was used to observe the influence of mm and recovery of hindlimb function injury in mice; and HE staining was used to observe the degree of injury caused by the pathological manifestations of spinal cord after spinal cord injury 8W; SCI was detected by HE staining. 3D 1W, 2W mouse spinal cord, 4W, spinal cord pathological features; GFAP immunohistochemical staining after SCI 3D, 1W, 2W, 4W, characteristics of mouse spinal cord astrocytes in response to 8W; to direct BDA The relationship between 8W nerve regeneration and glial scar after SCI in mice was observed by tracer. The activation characteristics of microglia / macrophages in 3D, 1W, 2W and 8W after SCI were detected by flow cytometry.
The third part: using the mouse spinal cord crush model in C3-/- mice and wild-type C3+/+ mice as experimental animal, we used the BMS score to assess hindlimb function, pathological changes of the spinal cord were observed by HE staining, flow cytometry detection of microglia / macrophages activation, expression of astrocyte GFAP immunohistochemistry and Western blot technology, comparative Observation on effects of C3 gene knockout on the functional recovery after SCI mice and explore its mechanism.
Result:
Part one:
1. compared with the PBS control group, the expression of IL-1 beta and TNF- alpha mRNA of 3,6,9,12h in microglia stimulated by 0.5 mg/ml serum albumin increased significantly.
2.0.1,0.5,1.0,2.0 mg/ml albumin after 24h stimulation of microglial cells in the culture supernatant of IL-1 beta, TNF- alpha concentration increased with increasing concentrations of serum albumin, 2 mg/ml stimulation group IL-1 beta, TNF- alpha was the highest; the albumin stimulation group IL-1 beta concentrations and TNF- concentrations were significantly higher than that of 0 mg/ml group; LPS group was significantly higher than that of no stimulation group.
After the action of serum albumin of 3.0.5 mg/ml, the secretion of IL-1 beta in microglia increased with time and increased at all time points after 6h. The secretion of TNF- alpha increased by time and increased at the beginning and after time of 3H.
4. the range and number of microglia activation caused by the injection of serum albumin in mice was significantly stronger than that in the PBS control group.
The second part:
1. successfully established stable C57BL/6J mouse spinal cord clamp injury animal model, BMS score showed hindlimb motor function after SCI mice have different degrees of injury, after 8W behavioral observations have been restored, but different injury degree of mice recovery were significantly different. Pinch the lighter (to 0.5mm), the better; crush the heavier (to 0.1mm), the worse the recovery.
The pathological changes of the spinal cord and the behavior of 2. different injury degree of mice 8W learning results. To clip the mice of 0.1mm spinal cord injury range widely, scar visible clip wound infiltration of inflammatory cells and the formation of tiny hole formation; with the weakening of crush degree, scar area gradually reduced, normal tissue residue gradually increased.
Observed continuous pathology in 3. 4W after injury: 3D after injury to spinal cord degeneration, necrosis, as the main performance; 1W has a large number of inflammatory cell infiltration, and a secondary injury area, visible damage on both sides of the edema and vacuolar 2W; there is still a large number of inflammatory cell infiltration, secondary injury and edema region is 1W reduced 4W; only the residual inflammatory cell infiltration, secondary injury decreased tissue into the repair period.
4. 8W after injury of astrocytes in continuous observation: 3D after injury, astrocytes had no obvious change. 1W; astrocyte hypertrophy, thickening of neurite growth were activated obviously, extends to the damage zone on both sides of head and tail; 2W the number of astrocytes was 1W but obviously increased, mast cells and processes the form as 7d; 4W and 8W activation degree and range of astrocytes decreased significantly, but in the middle of the scar tissue around the injury is wrapped with morphological hypertrophy, prominences reactive astrocytes glial scar formation, and a distance astrocyte morphology has been close to normal.
5.BDA anterograde tracing found SCI 10w+2d after crush injury to 0.25 mm mice in the spinal cord, the head side of the corticospinal tract most BDA markers to a wound, but cannot continue downward, forming a plurality of round ball in the final clip wound side head, only a small number of BDA nerve fibers in the dorsal lateral color original corticospinal tract position by clamping the wound, wound to clamp the caudal. After stained by GFAP, visible BDA final ball formation position exactly astrocyte hyperplasia formation of glial scar.
6. flow cytometry detection of CD68 positive cells suggested that the microglia / macrophages were activated and proliferated at 3D after injury, and the activation state was very strong at 3D. The number of microglia / macrophages increased to a peak at 7d and lasted until 8W after injury.
The third part:
The 1.BMS score showed that the recovery of hind limb function in C3-/- mice was better than that in group C3+/+.
2.HE staining showed that the infiltration of inflammatory cells in spinal cord injury site of 4W C3-/- mice after clipping was significantly less than that of C3+/+ group, while residual normal tissue was more than that of C3+/+ mice.
The detection of CD68 positive cells by 3. flow cytometry showed that the activation degree of microglia / macrophage in 3D, 7d and C3-/- mice after injury was significantly weaker than that of C3+/+ mice.
4. immunohistochemistry and Western blot showed that the expression of 4W GFAP in C3-/- mice was significantly weaker than that of C3+/+ mice.
Conclusion:
1., this study confirms that serum albumin can stimulate the activation and proliferation of microglia, and promote its secretion of IL-1 beta, TNF- alpha and proinflammatory cytokines, suggesting that serum albumin may play an important role in CNS inflammatory response.
2., a stable, easy to operate and reproducible mouse SCI model was established in this study. It is not only suitable for the study of microglia activation and inflammatory response in acute phase of SCI, but also for SCI chronic astrocytes forming glial scar and nerve regeneration.
The 3. complement system may increase the inflammatory response and promote the formation of astrocyte scar, which has an adverse effect on the repair of spinal cord injury in mice.
To sum up, through the study of serum albumin and complement, we further enrich the understanding of the role of serum factors in the inflammatory response after central nervous system injury, and provide a theoretical basis for the intervention of inflammatory response in the treatment of central nervous system diseases.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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