【摘要】：在中樞神經(jīng)系統(tǒng)(Central Nervous System,CNS)的創(chuàng)傷性脊髓損傷(Spinal Cord Injury,SCI)和腦卒中以后,損傷的軸突很難再生。外部環(huán)境和神經(jīng)元的內(nèi)在特性都是CNS軸突再生失敗的原因[1],內(nèi)因是成熟的神經(jīng)元已經(jīng)失去了再生潛能,外因?yàn)閾p傷后局部微環(huán)境不利于軸突的生長,外源性的抑制軸突的因子有髓鞘相關(guān)抑制分子,膠質(zhì)瘢痕,炎癥等[2]。決定軸突再生的內(nèi)源性因子有隨年齡變化的基因,轉(zhuǎn)錄因子,細(xì)胞骨架調(diào)節(jié)因子,線粒體等[3]。通過中和外源性的抑制分子如NOGO,髓鞘相關(guān)抑制蛋白(Myelin-associated Glycoprotein,MAG),軟骨素酶ABC(Chondroitinase ABC),免疫調(diào)節(jié)等改善軸突生長的外部壞境;用基因相關(guān)的技術(shù)調(diào)節(jié)如雷帕霉素靶蛋白(The Mammalian Target of Rapamycin,mTOR),信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄激活因子3(Signal Transducer and Activator of Transcription 3,STAT3),Kruppel樣因子(Kruppel-like Factors,KLFs),b-Raf激酶(Raf Kinase,b-Raf)和SOX11等信號(hào)通路,聯(lián)合腦源性神經(jīng)營養(yǎng)因子(Brain-derived Neurotrophic Factor,BDNF),睫狀神經(jīng)生長因子(Ciliary Neurotrophic Factor,CNTF),胰島素樣生長因子1(Insulin-like Growth Factor 1,IGF1),能很好的促進(jìn)CNS軸突再生,但能應(yīng)用于臨床的治療方法極少。骨橋蛋白(Osteopontin,OPN)最初在骨基質(zhì)中發(fā)現(xiàn),是一個(gè)可溶性的蛋白,后來研究發(fā)現(xiàn)它可以作為一個(gè)細(xì)胞因子廣泛存在于各個(gè)器官,能夠和細(xì)胞表面的整合素(Integrins)以及CD44結(jié)合,向細(xì)胞內(nèi)傳遞信號(hào),激活激酶級(jí)聯(lián)反應(yīng)和轉(zhuǎn)錄因子,促進(jìn)細(xì)胞的粘附,運(yùn)動(dòng)和生存[4]。我們前期的研究發(fā)現(xiàn),聯(lián)合應(yīng)用OPN/IGF1/CNTF,能夠促進(jìn)視神經(jīng)損傷后的軸突再生[5,6];腹腔注射增加軸突傳導(dǎo)速率的鉀離子通道阻斷劑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),能夠促使損傷動(dòng)物的視覺敏感度明顯提高[5]。但是在SCI和腦卒中模型中,OPN/IGF1是否能夠促進(jìn)軸突再生,進(jìn)而促進(jìn)動(dòng)物運(yùn)動(dòng)功能恢復(fù)還未可知。程序性死亡1(Programed Death-1,PD-1)是一個(gè)I型跨膜蛋白,由一個(gè)IgV樣的胞外段,一個(gè)跨膜區(qū)域和胞內(nèi)段組成。PD-1的胞內(nèi)段分兩部分,免疫受體酪氨酸相關(guān)的抑制性基序(Immunoreceptor Tyrosine-based Inhibitory Motif,ITSM)和免疫受體酪氨酸相關(guān)的轉(zhuǎn)化基序(Immunoreceptor Tyrosine-based Switch Motif,ITSM)[7]。PD-1屬于CD28家族成員之一[8]。它的受體(PD-L1/PD-L2)廣泛表達(dá)在T細(xì)胞,B細(xì)胞,自然殺傷性T細(xì)胞(Natural Killer T cell,NKT),巨噬細(xì)胞和樹突狀細(xì)胞(Dendritic Cells,DC)等[9,10]。在T細(xì)胞中,PD-1通過和它的受體相互作用,能夠抑制細(xì)胞增殖和細(xì)胞因子的分泌[11,12]。在B細(xì)胞中,PD-1能夠抑制B細(xì)胞的激活,擴(kuò)增和抗體合成[13]。用PD-1敲除的小鼠研究發(fā)現(xiàn),PD-1是免疫反應(yīng)的負(fù)性調(diào)節(jié)分子,在中樞和外周耐受中都發(fā)揮重要作用[10,14]。腦卒中以后小膠質(zhì)/巨噬細(xì)胞表達(dá)PD-1,和野生小鼠相比,PD-1敲除的小鼠梗死體積變大[15]。我們前期的研究發(fā)現(xiàn),SCI后,和野生小鼠相比,PD-1敲除的小鼠運(yùn)動(dòng)功能恢復(fù)差,體內(nèi)、外實(shí)驗(yàn)都顯示PD-1敲除以后促進(jìn)巨噬細(xì)胞向M1型極化。在本實(shí)驗(yàn)中的動(dòng)物模型中,我們建立兩種模型,錐體束半切模型(Pyramidotomy,PY)和光化學(xué)栓塞腦卒中模型,在損傷的對(duì)側(cè)皮層注射AAV-mcherry和AAV-OPN/IGF1或者AAV-PLAP,每隔一周進(jìn)行行為學(xué)檢測(cè)、錄像,行為學(xué)檢測(cè)方法有糖丸取回(Single-pallet Retrieval),膠帶移除(Sticker Remove),吃意面(Pasta Handling),不規(guī)律水平步行梯(Irregular Ladder Walking)等。損傷后13周腹腔注射4-AP-3-MeOH,檢測(cè)相關(guān)行為學(xué)變化。接著消除OPN/IGF1小鼠部分發(fā)芽的神經(jīng)元后,繼續(xù)進(jìn)行相關(guān)的行為學(xué)檢測(cè)。最后在損傷20周動(dòng)物灌注取材,進(jìn)行免疫組化染色,分析未損傷軸突向?qū)?cè)發(fā)芽的情況。體外實(shí)驗(yàn),通過培養(yǎng)小膠質(zhì)/巨噬細(xì)胞,給予脂多糖(Lipopolysaccharide,LPS)和γ干擾素(Interferon-γ,IFN-γ)刺激后,流式檢測(cè)PD-1及其受體PD-L1/PD-L2的表達(dá)情況。接著分別培養(yǎng)野生小鼠,PD-1敲除小鼠,PD-1高表達(dá)小鼠和PD-L1敲除小鼠來源的巨噬細(xì)胞,給予LPS和IFN-γ刺激不同時(shí)間,用Western Blot,分子克隆,藥物阻斷等方法,研究分析PD-1調(diào)節(jié)巨噬細(xì)胞極化的分子機(jī)制。在PY模型中,行為學(xué)恢復(fù)方面,實(shí)驗(yàn)組和對(duì)照組沒有明顯區(qū)別;但皮層注射AAV-OPN/IGF1的頸段脊髓軸突代償性的發(fā)芽明顯多于AAV-PLAP組。直徑4mm的光化學(xué)栓塞模型中,Sticker remove表現(xiàn)為自發(fā)性恢復(fù);Single-pallet retrieval幾乎沒有恢復(fù);在損傷后的第十周,實(shí)驗(yàn)組Irregular ladder walking恢復(fù)好于對(duì)照組,有統(tǒng)計(jì)學(xué)差異。脊髓未損傷側(cè)軸突向?qū)?cè)發(fā)芽情況與PY類似。接著我們做了直徑為2.5mm的光化學(xué)栓塞模型,同樣在損傷對(duì)側(cè)皮層注射AAV-OPN/IGF1或者AAV-PLAP,進(jìn)行行為學(xué)分析發(fā)現(xiàn),在損傷后第八周實(shí)驗(yàn)組Food retrieval和Irregular ladder walking的恢復(fù)情況明顯好于對(duì)照組。小鼠腹腔注射4-AP-3-MeOH以后,AAV-PLAP組行為學(xué)無變化,而AAV-OPN/IGF1的小鼠運(yùn)動(dòng)行為學(xué)有了更好的恢復(fù)。實(shí)驗(yàn)組和對(duì)照組在損傷的體積方面沒有差異,但是在皮層,皮層下的各個(gè)層面及脊髓頸段和腰段,AAV-OPN/IGF1組的軸突發(fā)芽數(shù)量和標(biāo)記軸突的熒光強(qiáng)度都多于AAV-PLAP組。消除OPN/IGF1組小鼠發(fā)芽至損傷側(cè)的神經(jīng)元后,OPN/IGF1引起的Single-pallet retrieval和Irregular ladder walking的行為學(xué)恢復(fù)也消失;解剖學(xué)分析頸段和腰段發(fā)芽的軸突數(shù)量也相應(yīng)的減少。體外培養(yǎng)腹腔來源的巨噬細(xì)胞,巨噬細(xì)胞系Raw264.7,骨髓干細(xì)胞誘導(dǎo)的巨噬細(xì)胞和新生鼠皮層小膠質(zhì)細(xì)胞發(fā)現(xiàn),在LPS+IFN-γ的作用下,小膠質(zhì)/巨噬細(xì)胞能夠表達(dá)PD-1及其受體PD-L1,但不表達(dá)PD-L2;在LPS+IFN-γ作用不同時(shí)間后,和野生型巨噬細(xì)胞相比,PD-1和PD-L1敲除的巨噬細(xì)胞,誘導(dǎo)型一氧化氮合酶(Inducible Nitric Oxide Synthase,i NOS)表達(dá)增高,流式檢測(cè)腫瘤壞死因子(Tumor Necrosis Factor alpha,TNF-α)陽性的細(xì)胞比例也增高,高表達(dá)PD-1的巨噬細(xì)胞表達(dá)iNOS降低;敲除PD-1和敲除PD-L1的巨噬細(xì)胞,Stat1,p-Stat1和p-NF-κB表達(dá)升高,蛋白激酶B(Protein Kinase B,PKB/Akt)/mTOR信號(hào)通路激活增加,高表達(dá)PD-1以后,Stat1和NF-κB蛋白表達(dá)并沒有低于野生型,但mTOR下核糖體蛋白S6激酶(Ribosomal Protein S6 Kinase,S6K)活性明顯降低。野生型和PD-1敲除的腹腔巨噬細(xì)胞,在給予LPS+IFN-γ的同時(shí)用不同濃度的雷帕霉素阻斷mTOR,發(fā)現(xiàn)敲除PD-1的巨噬細(xì)胞iNOS表達(dá)明顯低于WT巨噬細(xì)胞,說明PD-1敲除可能通過Akt/mTOR信號(hào)通路促進(jìn)巨噬細(xì)胞向M1型極化。通過以上實(shí)驗(yàn),說明應(yīng)用OPN/IGF1能夠促進(jìn)CNS損傷后皮質(zhì)脊髓束(The Corticospinal Tract,CST)的發(fā)芽,進(jìn)而促進(jìn)動(dòng)物運(yùn)動(dòng)功能的恢復(fù);敲除PD-1以后,可能通過激活A(yù)kt/mTOR信號(hào)通路促進(jìn)巨噬細(xì)胞向M1型極化。我們的研究為以后臨床應(yīng)用OPN/IGF1促進(jìn)軸突再生和了解PD-1調(diào)節(jié)巨噬細(xì)胞極化的分子機(jī)制提供了實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R741
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本文編號(hào)：2231098