阿托伐他汀修饰的树突状细胞对实验性自身免疫性神经炎的治疗研究

发布时间：2018-05-01 18:48

  本文选题：阿托伐他汀 + 树突状细胞　； 参考：《山东大学》2014年博士论文

【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。
[Abstract]:Background: human Green Barre syndrome (GBS) is a common cause of neuromuscular paralysis, and acute inflammatory demyelinating polyneuropathy (AIDP) is the most common type of autoimmune disease, which is mainly involved in the peripheral nervous system (PNS), and the pathogenesis is mainly mediated by CD4+T cells. Sexual autoimmune neuritis (EAN) is a recognized animal model for the study of human AIDP. In susceptible animals, the immunization of the same antigen in the peripheral nervous system (such as BPM, PO, etc.) can induce the occurrence of EAN in susceptible animals.
Dendritic cells (DCs), a classic antigen presenting cell, can initiate and regulate immune responses. Compared with activated DCs, immature DCs, characterized by its downregulation of surface CO stimulators such as CD80, CD86 and MHC- II, is used in Immunoregulation by multiple effect mechanisms, and it can induce the low responsiveness of T cells. This is a present phenomenon. Elephants have been used to control some autoimmune diseases, such as type 1 diabetes (type-I diabetes) and rheumatoid arthritis (RA). In the past twenty years, researchers have tried to create DCs that can induce immune tolerance in different ways, collectively called "tolerance DCs", which are mostly used in animal models, including experiments. Autoimmune autoimmune cerebrospinal meningitis (EAE), experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune uveitis (EAU).
There are many ways to produce tolerable DCs in vitro, the most common and effective use of immunosuppressive agents, such as the use of IL-10, TGF- beta, and the use of immunosuppressive drugs to regulate the function of DC and to induce tolerance DCs production. HMG-CoA reductase (a key enzyme in the process of cholesterol synthesis) is competitively suppressed in the metholate pathway, which is widely used in the treatment of atherosclerotic diseases and hyperlipidemia. Studies have shown that statins have immunoregulation and anti-inflammatory effects, especially the differentiation and maturation of DCs. It was found that atorvastatin could inhibit the maturation of spleen derived DCs, and the expression of CD80 and CD86 in the surface synergistic stimulator decreased significantly, and the statins modified DCs could reduce the symptoms of EAMG and the inflammation in the body, which showed the up regulation of the regulatory T cells (Treg cells) and the transformation of Th1/ Th17 cytokines to Th2 cytokine.
Statins modified DCs has no immunoregulation effect on EAN or GBS. In this study, we use atorvastatin to modify DCs to induce immune tolerance in this study. At the onset of the disease, the DCs was used to intervene in EAN rats, and the results of atorvastatin modified DCs were found. It has protective effect on EAN rats. The mechanism is mainly related to the up regulation of the number of Treg cells in the peripheral lymph nodes and the number of Foxp3 positive cells in the thymus, the up regulation of the number of NK and NKT cells in the peripheral lymph nodes, the decrease of Thl/Th17 type cytokines, the decrease of inflammatory cells in the peripheral nervous system and the inhibition of the proliferation of lymphocytes.
Objective: To investigate the therapeutic effect of atorvastatin modified DCs on experimental autoimmune neuritis and its immunomodulatory mechanism.
Research methods:
1. the EAN model was established and the clinical evaluation of BPM was established. The antigen was completely dissolved in IFA and H37Ra strains of Mycobacterium tuberculosis. The fully mixed antigen was injected subcutaneously in the rat's double foot pad. The dosage of each rat was 200 mu L, the animal model was immunized for zeroth days, and the symptoms of rats were carried out every day. Observation and clinical evaluation (double blind method) until fourteenth days after immunization.
2. the preparation and identification of the tolerance DCs of the spleen of healthy Lewis rats (attention aseptic operation), by grinding to prepare the mononuclear cell suspension. After breaking the red cell membrane, under the condition of 37, 5%CO2, the cell liquid in the culture bottle will be cultured 2h. abandoned suspension cell, leaving the adherent cell, and adding complete culture to continue to cultivate.18 under the above conditions. Ato Vatatin (final concentration of 10 mu M) dissolved in two methyl sulfoxide (DMSO) was added into the culture bottle, and the suspended cells were labeled as statin modified DCs (statin-DCs) and unstatin modified DCs (untreated-DCs) in the culture bottle with equal volume DMSO. culture in the contrast culture bottle, and the CD80, CD86, and CD86 were detected by flow cytometry. HC-II molecule.
3. group and intervention of 3. animals were randomly divided into three groups, five in each group, five in each group. In the fifth day of immunization, the treatment group was given a statin modified DCs, without a statin modified DCs intraperitoneal injection, and the number of cells injected in each rat was 1 * 106, and the control group (control group) was given the equal volume of 1640 medium in the abdominal cavity.
4. to prepare the lymphoid mononuclear cells (MNC) to kill the rats at the peak of EAN, take the inguinal lymph nodes, grind the lymph nodes, prepare the MNC and count the lymph nodes under the aseptic condition, and adjust the cell concentration to 2 x 106 cells, /ml.
5. flow cytometry was used to detect CD80, CD86 and MHC- II on the surface of lymph node mononuclear cells, and MNC was taken from the lymph nodes. After washing, FITC labeled mice CD86 and MHC-II antibodies were added to the mice, and the CD80 antibody of mice marked with PE marked, and the steps were incubated, suspended and filtered, and the flow cytometry was used.
6. flow cytometry was used to detect the MNC of lymph nodes in NK and NKT cells. After washing, the mice with FITC labeled anti CD3 antibody and PE marked mouse CD161a antibody were incubated, suspended and filtered, and the flow cytometry was used.
7. flow cytometry was used to detect Th1/Th2/Th17 type cytokine MNC, after washing, fixing, and breaking membrane, adding PE labeled mice IL-10 antibody, FITC labeled anti rat IFN- gamma antibody, PE labeled anti rat TNF- a antibody and FITC labeled anti rat IL-17A, after incubation, suspension, filtration and other steps, flow cytometer detection.
The 8. flow cytometry was used to detect the lymph node MNC in Treg cells. After washing, the anti CD25 antibody (mouse anti rat) and FITC labeled anti CD4 antibody (mice anti rat) were added respectively after washing, and 30min was incubated under the condition of 4 degrees centigrade, and 30min was incubated to avoid light. After washing, the fixed / broken membrane working fluid was added and mixed, and the night was incubated at 4 degrees, and the detergent cells were added to the Foxp3 resistance of PE-Cy5 markers. The 30min was incubated at 4 C, suspended and filtered, and detected by flow cytometry.
9. lymphocyte proliferation experiment mixed the same group of cell suspension in the same proportion, adding 0.5 L CFSE, incubating 30min at 37 degrees centigrade, adding pre cold 1640, incubating 5min on ice, adding complete medium after centrifugation and adding the same cell to 24 hole plates, adding BPM or ConA, respectively, at 37, and 5%CO2 under the condition of 72h; The cells in the 24 orifice plate were collected in the flow tube, centrifuged 5min (1200rpm/min), and then mixed with the supernatant. After washing, the anti rat CD4 antibody labeled with PE was added, and the steps were incubated, suspended and filtered, and the flow cytometry was used.
10. the histopathological examination was at the peak of the onset of the disease, fourteenth days after the immunization. After the death of the rat, the sciatic nerve was taken, 10% polyoxymethylene was fixed, paraffin was embedded, and the routine section was sliced. The steps were followed by dewaxing, hydrating, hematoxylin staining, 0.5% eosin staining, dehydration, transparency and sealing. The infiltration of inflammatory cells in the sciatic nerve was observed under microscope. Number.
11. thymus immunohistochemical staining was used to detect the paraffin paraffin embedded section, routine dewaxing, hydration, citrate antigen repair, H202 elimination of endogenous peroxidase, adding dilution good first antibody, rat anti mouse / rat Foxp3,4 C overnight. After rewarming, HRP labeled sheep two resistance, 37 degrees centigrade incubated for 1h, DAB color after washing and Soviet wood after washing. The Foxp3+ cells in the thymus of rats were observed under microscope and counted.
The results of the study:
1. the effect of atorvastatin on the DC phenotype was significantly lower than that of the non statin modified DCs surface costimulator CD80 and CD86, but there was no significant difference in the MHC-II molecule.
2. the incidence and clinical score of the three groups of rats were compared with the control group. The statin modified DCs significantly relieved the clinical symptoms of EAN rats at eleventh to fourteenth days of immunization. Compared with the unmodified DCs, the statin modified DCs significantly alleviated the clinical symptoms of EAN rats in the twelfth to fourteenth days of immunization; the control group and the untreated group had not been repaired. There was no significant difference in clinical score between the DCs treatment group and the control group.
3. atorvastatin modified DCs therapy reduced inflammatory cells in the sciatic nerve of EAN rats to decrease the inflammatory cells in the sciatic nerve of the sciatic nerve of EAN rats compared with those in the untreated DCs treatment group and the control group.
4. atorvastatin modified DCs was used to reduce the expression of CD80, CD86 and MHC- II on the mononuclear cells of EAN rat lymph nodes. Compared with the control group, the CD80, CD86 and MHC-II in the lymph node mononuclear cells of the EAN rats were significantly reduced in the statin modified DCs group, and the above indexes were also lower than those in the untreated DCs treatment group. Moreover, there was no significant difference in CD80 and CD86 on the mononuclear cells of the lymph nodes in the control group and in the untreated DCs treatment group, but the MHC-II was significantly lower in the untreated DCs treatment group than in the control group.
5. statin modified DCs therapy reduced the expression of Thl and Thl7 type cytokines in the EAN rat lymph node mononuclear cells compared with the control group. The IFN- gamma, TNF- A and IL-17A in the lymphoid mononuclear cells of the EAN rats were significantly reduced in the statin modified DCs treatment group, and the IL-17A and TNF- alpha were lower than those in the non treatment DCs treatment group, but there was no significant difference. At the same time, compared with the control group, the IFN- gamma and TNF- alpha in the untreated DCs treatment group decreased significantly, while there was no significant difference in the IL-17A between the two groups, and there was no significant difference in IL-10 among the three groups.
6. statin modified DCs increased the number of NK and NKT cells in the lymph node mononuclear cells compared with the control group and the untreated DCs group, and the CD3-CD161a+NK cells were significantly increased in the statin modified DCs treatment group. Compared with the other groups, the CD3+CD161a+NKT cells in the statin modified DCs treatment group were also elevated, and there was a significant difference between the control group and the control group. There was no significant difference in sex difference between the DCs treatment group and the untreated group.
7. statin modified DCs increased the number of CD4+Foxp3+Treg cells in the lymph node mononuclear cells compared with the control group and the untreated DCs treatment group. The statin modified DCs increased the number of CD4+Foxp3+Treg cells in the lymph node mononuclear cells, but there was no significant difference in the number of CD4+Foxp3 +Treg cells between the other two groups.
8. statin modified DCs increased the number of Foxp3+ cells in the thymus, compared with the control group and the untreated DCs treatment group, the number of Foxp3+ cells in the thymus gland in the statin modified DCs group increased significantly, and the number of Foxp3+ cells in the other groups had no significant difference.
9. statin modified DCs therapy inhibited the lymphocyte proliferation response compared with the control group and the untreated DCs group. The statin modified DCs significantly inhibited the lymphocyte proliferation response, whether in the case of BPM antigen stimulation or by ConA antigen stimulation; we did not find the lymph finer between the control group and the untreated DCs treatment group. There were significant differences in cell proliferation.
Conclusion:
1. atorvastatin can induce DCs to be a tolerable DCs.
2. atorvastatin modified DCs intervention can relieve the symptoms of experimental autoimmune neuritis.
3. atorvastatin modified DCs can induce central (thymus) and peripheral (lymph node) immune tolerance in experimental autoimmune neuritis.
Significance锛，

本文编号：1830625