研究中枢神经系统损伤后轴突再生和功能恢复的治疗策略
发布时间：2018-09-08 16:44
【摘要】：在中枢神经系统(Central Nervous System,CNS)的创伤性脊髓损伤(Spinal Cord Injury,SCI)和脑卒中以后,损伤的轴突很难再生。外部环境和神经元的内在特性都是CNS轴突再生失败的原因[1],内因是成熟的神经元已经失去了再生潜能,外因为损伤后局部微环境不利于轴突的生长,外源性的抑制轴突的因子有髓鞘相关抑制分子,胶质瘢痕,炎症等[2]。决定轴突再生的内源性因子有随年龄变化的基因,转录因子,细胞骨架调节因子,线粒体等[3]。通过中和外源性的抑制分子如NOGO,髓鞘相关抑制蛋白(Myelin-associated Glycoprotein,MAG),软骨素酶ABC(Chondroitinase ABC),免疫调节等改善轴突生长的外部坏境;用基因相关的技术调节如雷帕霉素靶蛋白(The Mammalian Target of Rapamycin,mTOR),信号转导和转录激活因子3(Signal Transducer and Activator of Transcription 3,STAT3),Kruppel样因子(Kruppel-like Factors,KLFs),b-Raf激酶(Raf Kinase,b-Raf)和SOX11等信号通路,联合脑源性神经营养因子(Brain-derived Neurotrophic Factor,BDNF),睫状神经生长因子(Ciliary Neurotrophic Factor,CNTF),胰岛素样生长因子1(Insulin-like Growth Factor 1,IGF1),能很好的促进CNS轴突再生,但能应用于临床的治疗方法极少。骨桥蛋白(Osteopontin,OPN)最初在骨基质中发现,是一个可溶性的蛋白,后来研究发现它可以作为一个细胞因子广泛存在于各个器官,能够和细胞表面的整合素(Integrins)以及CD44结合,向细胞内传递信号,激活激酶级联反应和转录因子,促进细胞的粘附,运动和生存[4]。我们前期的研究发现,联合应用OPN/IGF1/CNTF,能够促进视神经损伤后的轴突再生[5,6];腹腔注射增加轴突传导速率的钾离子通道阻断剂4-氨基吡啶(4-Aiminopyridine,4-AP)和它的衍生物4-氨基吡啶-3-甲醇(4-Aiminopyridine-3-Methanol,4-AP-3-MeOH),能够促使损伤动物的视觉敏感度明显提高[5]。但是在SCI和脑卒中模型中,OPN/IGF1是否能够促进轴突再生,进而促进动物运动功能恢复还未可知。程序性死亡1(Programed Death-1,PD-1)是一个I型跨膜蛋白,由一个IgV样的胞外段,一个跨膜区域和胞内段组成。PD-1的胞内段分两部分,免疫受体酪氨酸相关的抑制性基序(Immunoreceptor Tyrosine-based Inhibitory Motif,ITSM)和免疫受体酪氨酸相关的转化基序(Immunoreceptor Tyrosine-based Switch Motif,ITSM)[7]。PD-1属于CD28家族成员之一[8]。它的受体(PD-L1/PD-L2)广泛表达在T细胞,B细胞,自然杀伤性T细胞(Natural Killer T cell,NKT),巨噬细胞和树突状细胞(Dendritic Cells,DC)等[9,10]。在T细胞中,PD-1通过和它的受体相互作用,能够抑制细胞增殖和细胞因子的分泌[11,12]。在B细胞中,PD-1能够抑制B细胞的激活,扩增和抗体合成[13]。用PD-1敲除的小鼠研究发现,PD-1是免疫反应的负性调节分子,在中枢和外周耐受中都发挥重要作用[10,14]。脑卒中以后小胶质/巨噬细胞表达PD-1,和野生小鼠相比,PD-1敲除的小鼠梗死体积变大[15]。我们前期的研究发现,SCI后,和野生小鼠相比,PD-1敲除的小鼠运动功能恢复差,体内、外实验都显示PD-1敲除以后促进巨噬细胞向M1型极化。在本实验中的动物模型中,我们建立两种模型,锥体束半切模型(Pyramidotomy,PY)和光化学栓塞脑卒中模型,在损伤的对侧皮层注射AAV-mcherry和AAV-OPN/IGF1或者AAV-PLAP,每隔一周进行行为学检测、录像,行为学检测方法有糖丸取回(Single-pallet Retrieval),胶带移除(Sticker Remove),吃意面(Pasta Handling),不规律水平步行梯(Irregular Ladder Walking)等。损伤后13周腹腔注射4-AP-3-MeOH,检测相关行为学变化。接着消除OPN/IGF1小鼠部分发芽的神经元后,继续进行相关的行为学检测。最后在损伤20周动物灌注取材,进行免疫组化染色,分析未损伤轴突向对侧发芽的情况。体外实验,通过培养小胶质/巨噬细胞,给予脂多糖(Lipopolysaccharide,LPS)和γ干扰素(Interferon-γ,IFN-γ)刺激后,流式检测PD-1及其受体PD-L1/PD-L2的表达情况。接着分别培养野生小鼠,PD-1敲除小鼠,PD-1高表达小鼠和PD-L1敲除小鼠来源的巨噬细胞,给予LPS和IFN-γ刺激不同时间,用Western Blot,分子克隆,药物阻断等方法,研究分析PD-1调节巨噬细胞极化的分子机制。在PY模型中,行为学恢复方面,实验组和对照组没有明显区别;但皮层注射AAV-OPN/IGF1的颈段脊髓轴突代偿性的发芽明显多于AAV-PLAP组。直径4mm的光化学栓塞模型中,Sticker remove表现为自发性恢复;Single-pallet retrieval几乎没有恢复;在损伤后的第十周,实验组Irregular ladder walking恢复好于对照组,有统计学差异。脊髓未损伤侧轴突向对侧发芽情况与PY类似。接着我们做了直径为2.5mm的光化学栓塞模型,同样在损伤对侧皮层注射AAV-OPN/IGF1或者AAV-PLAP,进行行为学分析发现,在损伤后第八周实验组Food retrieval和Irregular ladder walking的恢复情况明显好于对照组。小鼠腹腔注射4-AP-3-MeOH以后,AAV-PLAP组行为学无变化,而AAV-OPN/IGF1的小鼠运动行为学有了更好的恢复。实验组和对照组在损伤的体积方面没有差异,但是在皮层,皮层下的各个层面及脊髓颈段和腰段,AAV-OPN/IGF1组的轴突发芽数量和标记轴突的荧光强度都多于AAV-PLAP组。消除OPN/IGF1组小鼠发芽至损伤侧的神经元后,OPN/IGF1引起的Single-pallet retrieval和Irregular ladder walking的行为学恢复也消失;解剖学分析颈段和腰段发芽的轴突数量也相应的减少。体外培养腹腔来源的巨噬细胞,巨噬细胞系Raw264.7,骨髓干细胞诱导的巨噬细胞和新生鼠皮层小胶质细胞发现,在LPS+IFN-γ的作用下,小胶质/巨噬细胞能够表达PD-1及其受体PD-L1,但不表达PD-L2;在LPS+IFN-γ作用不同时间后,和野生型巨噬细胞相比,PD-1和PD-L1敲除的巨噬细胞,诱导型一氧化氮合酶(Inducible Nitric Oxide Synthase,i NOS)表达增高,流式检测肿瘤坏死因子(Tumor Necrosis Factor alpha,TNF-α)阳性的细胞比例也增高,高表达PD-1的巨噬细胞表达iNOS降低;敲除PD-1和敲除PD-L1的巨噬细胞,Stat1,p-Stat1和p-NF-κB表达升高,蛋白激酶B(Protein Kinase B,PKB/Akt)/mTOR信号通路激活增加,高表达PD-1以后,Stat1和NF-κB蛋白表达并没有低于野生型,但mTOR下核糖体蛋白S6激酶(Ribosomal Protein S6 Kinase,S6K)活性明显降低。野生型和PD-1敲除的腹腔巨噬细胞,在给予LPS+IFN-γ的同时用不同浓度的雷帕霉素阻断mTOR,发现敲除PD-1的巨噬细胞iNOS表达明显低于WT巨噬细胞,说明PD-1敲除可能通过Akt/mTOR信号通路促进巨噬细胞向M1型极化。通过以上实验,说明应用OPN/IGF1能够促进CNS损伤后皮质脊髓束(The Corticospinal Tract,CST)的发芽,进而促进动物运动功能的恢复;敲除PD-1以后,可能通过激活Akt/mTOR信号通路促进巨噬细胞向M1型极化。我们的研究为以后临床应用OPN/IGF1促进轴突再生和了解PD-1调节巨噬细胞极化的分子机制提供了实验依据。
[Abstract]:It is difficult to regenerate injured axons after traumatic spinal cord injury (SCI) and stroke in the central nervous system (CNS). Endogenous inhibitors of axon regeneration include age-related genes, transcription factors, cytoskeleton regulatory factors, mitochondria and so on. Myelin-associated Glycoprotein (MAG), chondroitinase ABC (Chondroitinase ABC), and immunomodulation improve the external environment of axonal growth; gene-related techniques regulate such as the Mammalian Target of Rapamycin (mTOR), signal transducer and Activator of transcription Transcription 3, STAT3, Kruppel-like factors (KLFs), b-Raf kinase (Raf Kinase, b-Raf) and SOX11 signaling pathways are combined with brain-derived neurotrophic factor (BDNF), ciliary nerve growth factor (CNTF), insulin-like growth factor 1 (Insulin-Growth Factor 1). Factor 1, IGF1, can promote the regeneration of CNS axons very well, but it can be used in clinical treatment very few. Osteopontin (OPN) was first found in bone matrix, is a soluble protein, but later studies found that it can be widely distributed as a cytokine in various organs, and can be used in cell surface integrins (Integrins). Our previous study found that OPN/IGF1/CNTF could promote axonal regeneration after optic nerve injury [5,6]; intraperitoneal injection of potassium channel blocker 4-ammonia increased axonal conduction rate. 4-Aiminopyridine (4-AP) and its derivative 4-aminopyridine-3-methanol (4-AP-3-MeOH) can significantly improve the visual sensitivity of injured animals [5]. But whether OPN/IGF1 can promote axonal regeneration in SCI and stroke models and thus promote motor function recovery is not known. Programed Death-1 (PD-1) is a type I transmembrane protein consisting of an IgV-like extracellular segment, a transmembrane region and an intracellular segment. The intracellular segment of PD-1 is divided into two parts, the immunoreceptor tyrosine-related inhibitory sequence (ITSM) and the immunoreceptor tyrosine-related transforming group. Immunoreceptor Tyrosine-based Switch Motif [7]. PD-1 is a member of the CD28 family [8]. Its receptor (PD-L1/PD-L2) is widely expressed in T cells, B cells, natural killer T cells (NKT), macrophages and dendritic cells (DC) [9,10]. Interaction can inhibit cell proliferation and cytokine secretion [11,12]. In B cells, PD-1 can inhibit the activation, amplification and antibody synthesis of B cells [13]. In PD-1 knockout mice, PD-1 is a negative regulator of immune response and plays an important role in central and peripheral tolerance [10,14]. The infarct size of PD-1 knockout mice was larger than that of wild mice [15].Our previous study found that after SCI, the motor function of PD-1 knockout mice was poorer than that of wild mice, and both in vivo and in vitro experiments showed that PD-1 knockout promoted the polarization of macrophages to M1 type. Two models, pyramidal tract hemisection (PY) and photochemical embolization (PEE) were established. AAV-mcherry and AV-OPN/IGF1 or AAV-PLAP were injected into the injured contralateral cortex. Behavioral tests were performed every other week. Video and behavioral tests included single-pallet retrieval, Sticker Remove, and eating. After 13 weeks of injury, 4-AP-3-MeOH was injected intraperitoneally to detect the related behavioral changes. After removing part of the germinated neurons of OPN/IGF1 mice, the related behavioral tests were continued. At last, the animals were perfused for 20 weeks and stained with immunohistochemistry. In vitro, the expression of PD-1 and its receptor PD-L1/PD-L2 was detected by flow cytometry after microglia/macrophages were cultured and stimulated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Macrophages derived from mice and PD-L1 knockout mice were stimulated by LPS and IFN-gamma for different time. The molecular mechanism of PD-1 regulating macrophage polarization was studied by Western Blot, molecular cloning and drug blockade. In the 4 mm diameter photochemical embolization model, Sticker remove showed spontaneous recovery; Single-pallet retrieval showed almost no recovery; at the tenth week after injury, Irregular ladder walking recovered better in the experimental group than in the control group. Then we made a photochemical embolization model with a diameter of 2.5mm and injected AAV-OPN/IGF1 or AAV-PLAP into the injured contralateral cortex. Behavioral analysis showed that the recovery of Food retrieval and Irregular ladder walking in the experimental group was significantly better than that in the control group at the eighth week after injury. After injection of 4-AP-3-MeOH, the behavior of AAV-PLAP group remained unchanged, while the motor behavior of AAV-OPN/IGF1 mice recovered better. There was no difference in the volume of injury between the experimental group and the control group, but in the cortex, subcortical layers and the cervical and lumbar segments of the spinal cord, the number of axonal buds and the fluorescence of axons labeled in the AAV-OPN/IGF1 group. After eliminating the neurons germinated to the injured side of OPN/IGF1 mice, the behavioral recovery of single-pallet retrieval and Irregular ladder walking induced by OPN/IGF1 disappeared, and the number of axons germinated in the cervical and lumbar segments was also decreased by anatomical analysis. Cell line Raw264.7, macrophages induced by bone marrow stem cells and neonatal rat cortical microglia found that microglia/macrophages could express PD-1 and its receptor PD-L1, but not PD-L2, under the action of LPS+IFN-gamma. After LPS+IFN-gamma treatment for different time, compared with wild macrophages, PD-1 and PD-L1 knocked out macrophages were induced. Inducible Nitric Oxide Synthase (iNOS) expression increased, the proportion of Tumor Necrosis Factor alpha (TNF-alpha) positive cells increased, the expression of iNOS decreased in macrophages with high expression of PD-1, and increased expression of Stat1, p-Stat1 and p-NF-kappa B in macrophages with PD-1 knockout and P-NF-kappa B knockout. Protein Kinase B (PKB / Akt) / mTOR signaling pathway activation increased. After high expression of PD-1, the expression of Stat 1 and NF-kappa B protein was not lower than that of wild-type, but the activity of ribosomal protein S6 kinase (S6K) was significantly decreased under mTOR. Wild-type and PD-1 knockout peritoneal macrophages were treated with LPS + IFN-gamma without LPS + IFN-gamma. The same concentration of rapamycin blocked mTOR, and found that the expression of iNOS in PD-1 knockout macrophages was significantly lower than that in WT macrophages, indicating that PD-1 knockout may promote the polarization of macrophages to M1 type through Akt/mTOR signaling pathway. The results of this study provide experimental evidence for the clinical application of OPN/IGF1 in promoting axonal regeneration and understanding the molecular mechanism of PD-1 regulating macrophage polarization.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R741
，
本文编号：2231098
[Abstract]:It is difficult to regenerate injured axons after traumatic spinal cord injury (SCI) and stroke in the central nervous system (CNS). Endogenous inhibitors of axon regeneration include age-related genes, transcription factors, cytoskeleton regulatory factors, mitochondria and so on. Myelin-associated Glycoprotein (MAG), chondroitinase ABC (Chondroitinase ABC), and immunomodulation improve the external environment of axonal growth; gene-related techniques regulate such as the Mammalian Target of Rapamycin (mTOR), signal transducer and Activator of transcription Transcription 3, STAT3, Kruppel-like factors (KLFs), b-Raf kinase (Raf Kinase, b-Raf) and SOX11 signaling pathways are combined with brain-derived neurotrophic factor (BDNF), ciliary nerve growth factor (CNTF), insulin-like growth factor 1 (Insulin-Growth Factor 1). Factor 1, IGF1, can promote the regeneration of CNS axons very well, but it can be used in clinical treatment very few. Osteopontin (OPN) was first found in bone matrix, is a soluble protein, but later studies found that it can be widely distributed as a cytokine in various organs, and can be used in cell surface integrins (Integrins). Our previous study found that OPN/IGF1/CNTF could promote axonal regeneration after optic nerve injury [5,6]; intraperitoneal injection of potassium channel blocker 4-ammonia increased axonal conduction rate. 4-Aiminopyridine (4-AP) and its derivative 4-aminopyridine-3-methanol (4-AP-3-MeOH) can significantly improve the visual sensitivity of injured animals [5]. But whether OPN/IGF1 can promote axonal regeneration in SCI and stroke models and thus promote motor function recovery is not known. Programed Death-1 (PD-1) is a type I transmembrane protein consisting of an IgV-like extracellular segment, a transmembrane region and an intracellular segment. The intracellular segment of PD-1 is divided into two parts, the immunoreceptor tyrosine-related inhibitory sequence (ITSM) and the immunoreceptor tyrosine-related transforming group. Immunoreceptor Tyrosine-based Switch Motif [7]. PD-1 is a member of the CD28 family [8]. Its receptor (PD-L1/PD-L2) is widely expressed in T cells, B cells, natural killer T cells (NKT), macrophages and dendritic cells (DC) [9,10]. Interaction can inhibit cell proliferation and cytokine secretion [11,12]. In B cells, PD-1 can inhibit the activation, amplification and antibody synthesis of B cells [13]. In PD-1 knockout mice, PD-1 is a negative regulator of immune response and plays an important role in central and peripheral tolerance [10,14]. The infarct size of PD-1 knockout mice was larger than that of wild mice [15].Our previous study found that after SCI, the motor function of PD-1 knockout mice was poorer than that of wild mice, and both in vivo and in vitro experiments showed that PD-1 knockout promoted the polarization of macrophages to M1 type. Two models, pyramidal tract hemisection (PY) and photochemical embolization (PEE) were established. AAV-mcherry and AV-OPN/IGF1 or AAV-PLAP were injected into the injured contralateral cortex. Behavioral tests were performed every other week. Video and behavioral tests included single-pallet retrieval, Sticker Remove, and eating. After 13 weeks of injury, 4-AP-3-MeOH was injected intraperitoneally to detect the related behavioral changes. After removing part of the germinated neurons of OPN/IGF1 mice, the related behavioral tests were continued. At last, the animals were perfused for 20 weeks and stained with immunohistochemistry. In vitro, the expression of PD-1 and its receptor PD-L1/PD-L2 was detected by flow cytometry after microglia/macrophages were cultured and stimulated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Macrophages derived from mice and PD-L1 knockout mice were stimulated by LPS and IFN-gamma for different time. The molecular mechanism of PD-1 regulating macrophage polarization was studied by Western Blot, molecular cloning and drug blockade. In the 4 mm diameter photochemical embolization model, Sticker remove showed spontaneous recovery; Single-pallet retrieval showed almost no recovery; at the tenth week after injury, Irregular ladder walking recovered better in the experimental group than in the control group. Then we made a photochemical embolization model with a diameter of 2.5mm and injected AAV-OPN/IGF1 or AAV-PLAP into the injured contralateral cortex. Behavioral analysis showed that the recovery of Food retrieval and Irregular ladder walking in the experimental group was significantly better than that in the control group at the eighth week after injury. After injection of 4-AP-3-MeOH, the behavior of AAV-PLAP group remained unchanged, while the motor behavior of AAV-OPN/IGF1 mice recovered better. There was no difference in the volume of injury between the experimental group and the control group, but in the cortex, subcortical layers and the cervical and lumbar segments of the spinal cord, the number of axonal buds and the fluorescence of axons labeled in the AAV-OPN/IGF1 group. After eliminating the neurons germinated to the injured side of OPN/IGF1 mice, the behavioral recovery of single-pallet retrieval and Irregular ladder walking induced by OPN/IGF1 disappeared, and the number of axons germinated in the cervical and lumbar segments was also decreased by anatomical analysis. Cell line Raw264.7, macrophages induced by bone marrow stem cells and neonatal rat cortical microglia found that microglia/macrophages could express PD-1 and its receptor PD-L1, but not PD-L2, under the action of LPS+IFN-gamma. After LPS+IFN-gamma treatment for different time, compared with wild macrophages, PD-1 and PD-L1 knocked out macrophages were induced. Inducible Nitric Oxide Synthase (iNOS) expression increased, the proportion of Tumor Necrosis Factor alpha (TNF-alpha) positive cells increased, the expression of iNOS decreased in macrophages with high expression of PD-1, and increased expression of Stat1, p-Stat1 and p-NF-kappa B in macrophages with PD-1 knockout and P-NF-kappa B knockout. Protein Kinase B (PKB / Akt) / mTOR signaling pathway activation increased. After high expression of PD-1, the expression of Stat 1 and NF-kappa B protein was not lower than that of wild-type, but the activity of ribosomal protein S6 kinase (S6K) was significantly decreased under mTOR. Wild-type and PD-1 knockout peritoneal macrophages were treated with LPS + IFN-gamma without LPS + IFN-gamma. The same concentration of rapamycin blocked mTOR, and found that the expression of iNOS in PD-1 knockout macrophages was significantly lower than that in WT macrophages, indicating that PD-1 knockout may promote the polarization of macrophages to M1 type through Akt/mTOR signaling pathway. The results of this study provide experimental evidence for the clinical application of OPN/IGF1 in promoting axonal regeneration and understanding the molecular mechanism of PD-1 regulating macrophage polarization.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R741
，
本文编号：2231098
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