【作者】 徐华；

【导师】 段瑞生；

【作者基本信息】 山东大学 ， 神经病学（专业学位）， 2014， 博士

【摘要】 研究背景：人类格林-巴利综合征(GBS)是引起神经肌肉麻痹的普遍原因,而急性炎症性脱髓鞘性多发性神经病(AIDP),其最常见的类型,为一种自身免疫性疾病,临床上主要累及外周神经系统(PNS),而发病机制主要由CD4+T细胞参与介导。实验性自身免疫性神经炎(EAN)是公认的研究人类AIDP的动物模型,在易感动物中,通过外周神经系统中同种抗原的免疫(如BPM、PO等)可以诱导EAN的发生。树突状细胞(DCs)是经典的抗原递呈细胞,能够启动和调控免疫反应,与活化的DCs相比,未成熟的DCs,特征是其表面共刺激分子如CD80、CD86和MHC-Ⅱ分子下调,在免疫调节中通过多种效应机制起作用,而且,它能够诱导T细胞的低反应性,这种现象已经被人们用于控制某些自身免疫性疾病的研究,比如说1型糖尿病(type-I diabetes)及类风湿关节炎(RA)。近二十年间,研究者们尝试用不同的方式创造能够诱导免疫耐受的DCs,统称为“耐受性DCs",这些研究大都应用于动物模型,包括实验性自身免疫性脑脊髓膜炎(EAE),实验性自身免疫性重症肌无力(EAMG)及实验性自身免疫性葡萄膜炎(EAU)等。在体外修饰产生耐受性DCs的方式很多,最常见有效的是应用免疫抑制剂,例如应用IL-10, TGF-β等,而利用免疫抑制药物对DC功能进行药理调节进而诱导耐受性DCs产生的方式也做过广泛研究。包括阿托伐他汀在内的他汀类药物,在胆固醇生物合成的甲羟戊酸途径中可以竞争性抑制HMG-CoA还原酶(胆固醇合成过程中的一种关键酶),临床上广泛用于治疗动脉粥样硬化性疾病和高脂血症。研究表明他汀类药物具有免疫调节和抗炎作用,尤其可以抑制DCs的分化和成熟,我们的前期试验发现：阿托伐他汀可以抑制脾源性DCs的成熟,其表面协同刺激分子CD80和CD86表达明显下降,而且经他汀修饰的DCs可以减轻EAMG的症状和体内炎症,表现为调节性T细胞(Treg cells)的上调,Th1/Th17型细胞因子向Th2型细胞因子的转变。他汀修饰的DCs对EAN或GBS有无免疫调节作用,到目前为止还没有相关报道。在本研究中,我们在体外通过阿托伐他汀修饰DCs使之具有诱导免疫耐受的功能,在发病的起始阶段,利用上述DCs对EAN大鼠给予干预,结果发现阿托伐他汀修饰的DCs对EAN大鼠有保护作用,其机制主要与外周淋巴结中Treg细胞数量和胸腺中Foxp3阳性细胞数量的上调、外周淋巴结中NK细胞及NKT细胞数目的上调、Thl/Th17型细胞因子的降低、外周神经系统炎性细胞浸润减少及淋巴细胞增殖的抑制等有关。研究目的：探讨阿托伐他汀修饰的DCs对实验性自身免疫性神经炎的治疗作用及免疫调节机制。研究方法：1.建立EAN模型并临床评估提取BPM,并完全溶于弗氏不完全佐剂(IFA)和H37Ra株结核分枝杆菌中以配制抗原,在大鼠双后足垫皮下注射上述充分混匀的抗原,每只大鼠用量为200μl,以免疫动物造模。免疫当天定为第0天,每天对大鼠的症状进行观察并临床评估(双盲法观察)直至免疫后第14天。2.耐受性DCs的制备及鉴定取健康Lewis大鼠的脾脏(注意无菌操作),通过研磨以制备单个核细胞悬液。破红细胞膜后,在37℃、5%CO2条件下,将置于培养瓶中的细胞液培养2h。弃悬浮细胞,留取贴壁细胞,加入完全培养基于上述条件下继续培养。18h后向培养瓶中加入溶于二甲基亚砜(DMSO)的阿托伐他汀(终浓度10μM),对照培养瓶中加入等体积的DMSO。培养48h后收集悬浮的细胞分别标记为他汀修饰的DCs (statin-DCs)和未经他汀修饰的DCs(untreated-DCs)。通过流式检测不同组DCs表面CD80、 CD86、 MHC-II分子。3.动物分组及干预将EAN大鼠随机分为三组,每组五只,在免疫第五天,治疗组分别给予他汀修饰的DCs、未经他汀修饰的DCs腹腔注射,每只大鼠注射的细胞数为1×106个,对照组(control group)给予等体积的1640培养基腹腔注射。4.制备淋巴结单个核细胞(MNC)在EAN发病高峰期处死大鼠,在无菌条件下取其腹股沟淋巴结,研磨淋巴结,制备MNC并计数,将细胞浓度调整为2×106个细胞/ml。5.流式细胞仪检测淋巴结单个核细胞表面的CD80、 CD86及MHC-Ⅱ取淋巴结MNC,洗涤后分别加入FITC标记的小鼠抗大鼠CD86和MHC-II抗体,以及PE标记的小鼠抗大鼠CD80抗体,经孵育、重悬、过滤等步骤,流式细胞仪检测。6.流式检测NK和NKT细胞取淋巴结MNC,洗涤后分别加入FITC标记的小鼠抗大鼠CD3抗体和PE标记的小鼠抗大鼠CD161a抗体,经孵育、重悬、过滤等步骤,流式细胞仪检测。7.流式检测Th1/Th2/Th17型细胞因子取淋巴结MNC,经洗涤、固定、破膜等步骤,分别加入PE标记的小鼠抗大鼠IL-10抗体、FITC标记的抗大鼠IFN-γ抗体、PE标记的抗大鼠TNF-α抗体和FITC标记的抗大鼠IL-17A抗体,经孵育、重悬、过滤等步骤,流式细胞仪检测。8.流式检测Treg细胞取淋巴结MNC,洗涤后分别加入PE标记的抗CD25抗体(小鼠抗大鼠)和FITC标记的抗CD4抗体(小鼠抗大鼠),4℃条件下孵育30min,避光,洗涤后加入固定／破膜工作液并混匀,4℃孵育过夜,避光。洗涤细胞后加入PE-Cy5标记的Foxp3抗体4℃孵育30min,重悬、过滤后,流式细胞仪检测。9.淋巴细胞增殖实验将同组细胞悬液以同样比例混合,加入0.5μl CFSE,37℃避光孵育30min,加入预冷的1640,置于冰上孵育5min；离心弃上清后加入完全培养基,以相同细胞数种于24孔板,分别加入刺激物BPM或ConA,置于37℃、5%CO2条件下培养72h；将24孔板中的细胞分别收集到流式管中,离心5min (1200rpm/min),弃上清后混匀；洗涤后加入PE标记的抗大鼠CD4抗体,经孵育、重悬、过滤等步骤,流式细胞仪检测。10.组织病理学检测在发病高峰期即免疫后第14天,处死大鼠后取坐骨神经,10%多聚甲醛固定,石蜡包埋,常规切片,依次经脱蜡、水化、苏木素染色、0.5%伊红液染色、脱水、透明、封片等步骤,显微镜下观察坐骨神经中炎性细胞的浸润情况并计数。11.胸腺免疫组化检测大鼠胸腺石蜡包埋后切片,常规脱蜡、水化,柠檬酸盐抗原修复,H202消除内源性过氧化物酶,滴加稀释好的一抗,大鼠抗小鼠/大鼠Foxp3,4℃过夜。复温后洗涤,滴加HRP标记的羊抗大鼠二抗,37℃孵育1h,洗涤后DAB显色,苏木素复染,脱水、透明、封片。显微镜下观察大鼠胸腺中Foxp3+细胞的情况并计数。研究结果：1.阿托伐他汀对DC表型的影响他汀修饰的DCs较未经他汀修饰的DCs表面共刺激分子CD80和CD86的表达明显降低,而MHC-II分子没有显著性差异。2.三组大鼠的发病情况与临床评分与对照组相比,他汀修饰的DCs在免疫的第11到第14天,明显缓解了EAN大鼠的临床症状；而与未经修饰的DCs相比,他汀修饰的DCs在免疫的第12到第14天,明显减轻了EAN大鼠的临床症状；对照组与未经他汀修饰的DCs治疗组的临床评分之间无明显差异。3.阿托伐他汀修饰的DCs治疗减少EAN大鼠坐骨神经中的炎性细胞他汀修饰的DCs治疗组中EAN大鼠坐骨神经中炎性细胞较未处理DCs治疗组及对照组明显减少。4.阿托伐他汀修饰的DCs治疗下调EAN大鼠淋巴结单个核细胞上CD80、 CD86和MHC-Ⅱ的表达。与对照组相比,他汀修饰DCs治疗组中EAN大鼠淋巴结单个核细胞上CD80、 CD86及MHC-II均显著降低；与未处理DCs治疗组相比,上述指标亦有降低,但是没有显著性差异。而且,淋巴结单个核细胞上CD80和CD86在对照组中和未处理DCs治疗组中没有显著性差异,但是MHC-II在未处理DCs治疗组中明显降低,与对照组相比。5.他汀修饰的DCs治疗降低了EAN大鼠淋巴结单个核细胞中Thl和Thl7型细胞因子的表达与对照组相比,他汀修饰DCs治疗组中EAN大鼠淋巴结单个核细胞中的IFN-γ, TNF-α和IL-17A均显著降低；与未处理DCs治疗组相比,IL-17A和TNF-α降低,但无显著性差异；同时与对照组相比,未处理DCs治疗组中IFN-γ和TNF-α显著下降,而IL-17A在这两组中无显著性差异；三组之中,IL-10没有显著性差异。6.他汀修饰的DCs上调了淋巴结单个核细胞中NK和NKT细胞的数量与对照组和未处理DCs治疗组相比,CD3-CD161a+NK细胞在他汀修饰DCs治疗组明显升高；与另外两组相比,CD3+CD161a+NKT细胞在他汀修饰的DCs治疗组也有升高,而且与对照组之间有显著性差异,而与未处理DCs治疗组无显著性差异。7.他汀修饰的DCs上调了淋巴结单个核细胞CD4+Foxp3+Treg细胞的数量与对照组及未处理DCs治疗组相比,他汀修饰的DCs上调了淋巴结单个核细胞中CD4+Foxp3+Treg细胞的数量；而在其他两组之间未发现CD4+Foxp3+Treg细胞数量有显著性差异。8.他汀修饰DCs上调了胸腺中Foxp3+细胞的数量与对照组及未处理DCs治疗组相比,他汀修饰DCs治疗组中胸腺Foxp3+细胞的数量明显增加；另外两组中Foxp3+细胞数量无显著性差异。9.他汀修饰的DCs治疗抑制了淋巴细胞增殖反应与对照组及未处理DCs组相比,他汀修饰DCs显著抑制了淋巴细胞增殖反应,无论是在BPM抗原刺激的情况下,还是ConA抗原刺激的情况下；我们没有发现在对照组和未处理DCs治疗组之间,淋巴细胞增殖有显著性差异。结论：1.阿托伐他汀可诱导DCs成为耐受性DCs。2.阿托伐他汀修饰的DCs干预,可以减轻实验性自身免疫性神经炎的症状。3.阿托伐他汀修饰的DCs在实验性自身免疫性神经炎中可诱导中枢(胸腺)和外周(淋巴结)免疫耐受。意义：本研究通过阿托伐他汀诱导DCs成为耐受性DCs,给予EAN大鼠腹腔注射,显示他汀诱导的耐受性DCs能够减轻EAN的症状并诱导免疫耐受,这种作用主要是通过抑制淋巴结单个核细胞表面共刺激分子(CD80和CD86)及MHC-Ⅱ的表达、降低Thl/Th17型细胞因子、上调淋巴结中Treg细胞及胸腺中Foxp3细胞的表达及上调淋巴结单个核细胞中NK及NKT细胞数量实现的。本研究为人类GBS的治疗提供了新的思路。更多还原

【Abstract】 Background:Acute inflammatory demyelinating polyneuropathy (AIDP), the most common form of Guillain-Barre syndrome (GBS), is a CD4+T cell-mediated disease of the peripheral nervous system (PNS), with an annual incidence of0.62-2.66cases per100,000population. Experimental autoimmune neuritis (EAN) serves as an animal model for AIDP in humans, which can be induced in susceptible animal species and strains by immunization with an autoantigen emulsified in complete Freund’s adjuvant.Dendritic cells (DCs), being professional antigen-presenting cells, initiate and orchestrate immune responses. In contrast to activated DCs, immature DCs with low levels of major histocompatibility complex (MHC) and co-stimulatory molecules have been implicated in the regulation of immune responses through diverse effector mechanisms. Particularly, immature DCs are able to induce a state of hyporesponsiveness in T cells, and the phenomenon has been used for the control or suppression of the immune response. Therefore, some attempts have been made to modify DCs to retain their immature phenotype for the induction of antigen-specific tolerance and protect from some autoimmune diseases. Statins, including atorvastatin, are competitive inhibitors of HMG-CoA reductase that regulates cholesterol synthesis in the mevalonatepathway. They are widely used in the treatment of atherosclerosis and hypercholesterolemia. Clinical and experimental evidences have demonstrated that statins have pleiotropic effects that include anti-inflammatory and immunomodulatory effects, especially inhibiting DCs differentiation and maturation. Previously, our group found that atorvastatin suppressed the maturation of spleen derived DCs, on which with the lower expression of CD80and CD86, and the statin-modified DCs could ameliorate experimental autoimmune myasthenia gravis by up-regulated Treg cells and shifted Th1/Th17to Th2cytokines. So we presume that statin-modified DCs could also attenuate EAN by immune regulation. In the present study, we transferred statin-modified DCs to EAN rats in the initial phase of the disease to evaluate its possible role and immunomodulatory effects during the process.Objective:The aim of the present study was to analyze the effects of DCs modified with atorvastatin (statin-DCs) on the immune tolerance induction in Lewis rats with EAN.Methods:1. Induction of EAN and assessment of clinical symptoms200μl of the inoculum contained5mg BPM and2mg Mycobacterium tuberculosis emulsified in incomplete Freund’s adjuvant was subcutaneous injected into both hind footpads of the rats to induce EAN. Clinical scores were assessed immediately before immunization (day0) and thereafter every day until day14post immunization (p.i.).The severity of clinical symptoms was scored as follows:0=no illness;1=flaccid tail;2=dragging both hind limbs;3=paralysis of both hind limbs; and4=paralysis of four limbs or death, intermediate scores of0.5increment were given to rats with intermediate signs. The rats were monitored for clinical symptoms of disease by two independent investigators.2. DCs preparation, modification and injection DCs were prepared by the method of Duan et al. with some modifications. The spleens were removed from normal Lewis rats of6-8weeks old under aseptic conditions. Grinding the spleen from individual rats through cell strainers in medium to obtain the mononuclear cells (MNC) suspensions, then erythrocytes were lysed osmotically. DCs were further enriched by differential adherence by incubating cells. After removing the non-adherent cells, new RPMI1640medium containing gentamicin, penicillin and10%fetal bovine serum were added to the flasks. After18h of incubation, atorvastatin dissolved in DMSO was added to the flasks. The same volume of DMSO was added to the other flasks. After incubation, floating cells were collected as a DC-enriched fraction. Then statin-DCs and untreated-DCs were intraperitoneally transferred into EAN rats respectively on day5p.i. And the same volume of medium was transferred into the control rats. For phenotypic analysis, statin-DCs and untreated-DCs were washed with0.5%bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and stained with fluorescein isothiocyanate (FITC)-conjugated anti-rat MHC class Ⅱ, phycoerythrin (PE)-conjugated anti-rat CD80and FITC-conjugated anti-rat CD86antibodies, after resuspending, the cells were analyzed by FACS.3. Preparation of lymph node MNC Inguinal lymph nodes were removed under aseptic conditions when rats were sacrificed on day14p.i. MNC suspensions were obtained by grinding the organs through cell strainers in serum-free medium. Then cells were rediluted to a cell concentration of2*106cells/ml for all experiments.4. Flow cytometric analysis of surface molecules, NK and NKT cells, intracellular cytokines and Treg cells in MNC from lymph nodes MNC suspensions were incubated with PE-labeled anti-rat CD80, FITC-labeled anti-rat CD86, FITC-labeled anti-rat MHC class Ⅱ, FITC-labeled anti-rat CD3, and PE-labeled anti-rat CD161a in the dark, respectively; Lymph node MNC were fixed and permeabilized, then incubated with the PE-labeled IL-10, FITC-labeled IFN-γ, PE-labeled TNF-a and FITC-labeled IL-17A, in the dark respectively; Fixation and permeabilization of lymph node MNC were performed using the eBioscience Foxp3Staining Buffer Set. Then the cells were re-suspended in PBS respectively and analyzed by FACS.5. Analysis of cell viability MNC of rats in the same group were mixed at equally number and incubated with CFSE in the dark, then stained by adding ice-cold1640and incubated on ice, after concentration and suspension, the mentioned cells suspended in1000μl aliquots containing1x106cells were cultured in triplicates in flat-bottomed24-well microtiter plates in the presence of BPM (15μg/ml) or Concanavalin A (ConA,5μg/ml). After incubation in a humidified atmosphere of95%air and5%CO2at37℃, the cells were collected respectively in the tube and incubated with PE-labeled anti-rat CD4antibody in the dark. Lastly, the cells were washed and re-suspended in PBS. Then the cells were analyzed by FACS.6. Histopathological assessment After the rats killed on day14p.i. Sciatic nerve segments were excised close to the lumbar spinal cord, then fixed in10%paraformaldehyde and embedded in paraffin. Multiple longitudinal sections of sciatic nerves were stained with hematoxylin and eosin for evaluation of the inflammatory cells by light microscopy.7. Immunohistochemistry Thymuses obtained from the rats on day14p.i. were excised and fixed in10%paraformaldehyde, then embedded in paraffin. Paraffin tissue sections were deparaffinized and hydrated. And in order to block the endogenous peroxidase activity, the sections were treated with0.3%hydrogen peroxide. After antigen retrieval and incubated with rat anti-mouse/rat foxp3antibody, the sections were stained with HRP-conjugated goat anti-rat secondary antibody, followed by development with diaminobenzidine (DAB) substrate to detect the number of Foxp3+cells. As negative controls for immunostaining, the primary antibodies were omitted.8. Statistical analysis The statistics were calculated and evaluated by the SPSS17.0computer program. Differences among different groups were tested by one-factor analysis of variance (ANQVA) followed by Least Significant Difference (LSD) test as a post-hoc test. Results were presented as mean±SD and a level of p<0.05was considered significant.Results:1. Effects of atorvastatin on the phenotype of DCs The results showed that the expression of CD80and CD86was inhibited on statin-DCs when compared with those on untreated-DCs.2. Statin-DCs suppress severity of clinical EAN The rats in the statin-DC group exhibited lower clinical scores when compared with the rats in control group and untreated-DC group. While it has no significant difference of the clinical scores between untreated-DC group and control group. On the day of the experiment termination, the clinical score of the rats in the statin-DC group averaged1.55±0.45, meanwhile, the clinical scores in the control and the untreated-DCs group averaged2.7±0.57and2.4±0.42.3. Statin-DCs decrease the number of inflammatory cells in the PNS The rats in statin-DC treated group had fewer inflammatory cells in sciatic nerves than the rats in the untreated-DC and control group.4. Statin-DCs inhibit the expression of co-stimulatory molecules and MHC class II in MNC from lymph nodes The expression of CD80, CD86and MHC class II in lymphocytes was inhibited in the statin-DC group when compared with those in the control group and untreated-DC group but without significant difference. Moreover, the expression of CD80and CD86in lymphocytes between the control and the untreated-DC groups did not differ significantly, but the expression of MHC class II was lower in the untreated-DC group when compared with that in the control group.5. Statin-DCs decrease the Th1and Th17cytokines in MNC from lymph nodes IFN-y, TNF-a and IL-17A were decreased in the statin-DC group when compared with those in the control group. IL-17A and TNF-a were decreased in the statin-DC group compared with those in the untreated-DC group, however there were no significant differences. At the same time, IFN-y and TNF-a were lower in the untreated-DC group when compared with those in control group, and there was no significant difference of IL-17A between these two groups. There was no statistical difference for IL-10among the three groups.6. Statin-DCs increase the numbers of NK and NKT cells in MNC from lymph nodes The percentage of CD3+CD161a+NK cells was increased in the statin-DC group when compared with that in the control and the untreated-DC group, and the percentage of CD3+CD161a+NKT cells was increased in the statin-DC group when compared with that in the control as well as the untreated-DC group but without any statistical difference.7. Statin-DCs increase the number of CD4+Foxp3+T cells in MNC of the lymph nodes Statin-DCs increased the percentage of CD4+Foxp3+T cells among lymph node MNC when compared with untreated-DCs and control.8. Statin-DCs increase the number of Foxp3+cells in the thymus The number of Foxp3+cells in thymus of statin-DCs treated rats was increased when compared with those in the other two groups. There was no significant difference between the control group and untreated-DCs group.9. Statin-DCs suppress lymphocyte proliferation in EAN Lymphocyte proliferation was significantly decreased in the statin-DC group compared with those in the control and untreated-DC group with BPM or without BPM. There was no significant difference between the control and untreated-DC group.Conclusions:1. DCs treated with atorvastatin (statin-DCs) can be induced into tolerogenic DCs.2. Administration of statin-DCs ameliorate the clinical symptoms in EAN.3. Statin-DCs induce both central (thymus) and peripheral immune tolerance (lymph node) in EAN.更多还原