本文選題：阿托伐他汀 + 樹突狀細(xì)胞��； 參考：《山東大學(xué)》2014年博士論文

【摘要】：研究背景：人類格林-巴利綜合征(GBS)是引起神經(jīng)肌肉麻痹的普遍原因,而急性炎癥性脫髓鞘性多發(fā)性神經(jīng)病(AIDP),其最常見的類型,為一種自身免疫性疾病,臨床上主要累及外周神經(jīng)系統(tǒng)(PNS),而發(fā)病機(jī)制主要由CD4+T細(xì)胞參與介導(dǎo)。實(shí)驗(yàn)性自身免疫性神經(jīng)炎(EAN)是公認(rèn)的研究人類AIDP的動物模型,在易感動物中,通過外周神經(jīng)系統(tǒng)中同種抗原的免疫(如BPM、PO等)可以誘導(dǎo)EAN的發(fā)生。 樹突狀細(xì)胞(DCs)是經(jīng)典的抗原遞呈細(xì)胞,能夠啟動和調(diào)控免疫反應(yīng),與活化的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分子下調(diào),在免疫調(diào)節(jié)中通過多種效應(yīng)機(jī)制起作用,而且,它能夠誘導(dǎo)T細(xì)胞的低反應(yīng)性,這種現(xiàn)象已經(jīng)被人們用于控制某些自身免疫性疾病的研究,比如說1型糖尿病(type-I diabetes)及類風(fēng)濕關(guān)節(jié)炎(RA)。近二十年間,研究者們嘗試用不同的方式創(chuàng)造能夠誘導(dǎo)免疫耐受的DCs,統(tǒng)稱為“耐受性DCs",這些研究大都應(yīng)用于動物模型,包括實(shí)驗(yàn)性自身免疫性腦脊髓膜炎(EAE),實(shí)驗(yàn)性自身免疫性重癥肌無力(EAMG)及實(shí)驗(yàn)性自身免疫性葡萄膜炎(EAU)等。 在體外修飾產(chǎn)生耐受性DCs的方式很多,最常見有效的是應(yīng)用免疫抑制劑,例如應(yīng)用IL-10, TGF-β等,而利用免疫抑制藥物對DC功能進(jìn)行藥理調(diào)節(jié)進(jìn)而誘導(dǎo)耐受性DCs產(chǎn)生的方式也做過廣泛研究。包括阿托伐他汀在內(nèi)的他汀類藥物,在膽固醇生物合成的甲羥戊酸途徑中可以競爭性抑制HMG-CoA還原酶(膽固醇合成過程中的一種關(guān)鍵酶),臨床上廣泛用于治療動脈粥樣硬化性疾病和高脂血癥。研究表明他汀類藥物具有免疫調(diào)節(jié)和抗炎作用,尤其可以抑制DCs的分化和成熟,我們的前期試驗(yàn)發(fā)現(xiàn)：阿托伐他汀可以抑制脾源性DCs的成熟,其表面協(xié)同刺激分子CD80和CD86表達(dá)明顯下降,而且經(jīng)他汀修飾的DCs可以減輕EAMG的癥狀和體內(nèi)炎癥,表現(xiàn)為調(diào)節(jié)性T細(xì)胞(Treg cells)的上調(diào),Th1/Th17型細(xì)胞因子向Th2型細(xì)胞因子的轉(zhuǎn)變。 他汀修飾的DCs對EAN或GBS有無免疫調(diào)節(jié)作用,到目前為止還沒有相關(guān)報道。在本研究中,我們在體外通過阿托伐他汀修飾DCs使之具有誘導(dǎo)免疫耐受的功能,在發(fā)病的起始階段,利用上述DCs對EAN大鼠給予干預(yù),結(jié)果發(fā)現(xiàn)阿托伐他汀修飾的DCs對EAN大鼠有保護(hù)作用,其機(jī)制主要與外周淋巴結(jié)中Treg細(xì)胞數(shù)量和胸腺中Foxp3陽性細(xì)胞數(shù)量的上調(diào)、外周淋巴結(jié)中NK細(xì)胞及NKT細(xì)胞數(shù)目的上調(diào)、Thl/Th17型細(xì)胞因子的降低、外周神經(jīng)系統(tǒng)炎性細(xì)胞浸潤減少及淋巴細(xì)胞增殖的抑制等有關(guān)。 研究目的：探討阿托伐他汀修飾的DCs對實(shí)驗(yàn)性自身免疫性神經(jīng)炎的治療作用及免疫調(diào)節(jié)機(jī)制。 研究方法： 1.建立EAN模型并臨床評估提取BPM,并完全溶于弗氏不完全佐劑(IFA)和H37Ra株結(jié)核分枝桿菌中以配制抗原,在大鼠雙后足墊皮下注射上述充分混勻的抗原,每只大鼠用量為200μl,以免疫動物造模。免疫當(dāng)天定為第0天,每天對大鼠的癥狀進(jìn)行觀察并臨床評估(雙盲法觀察)直至免疫后第14天。 2.耐受性DCs的制備及鑒定取健康Lewis大鼠的脾臟(注意無菌操作),通過研磨以制備單個核細(xì)胞懸液。破紅細(xì)胞膜后,在37℃、5%CO2條件下,將置于培養(yǎng)瓶中的細(xì)胞液培養(yǎng)2h。棄懸浮細(xì)胞,留取貼壁細(xì)胞,加入完全培養(yǎng)基于上述條件下繼續(xù)培養(yǎng)。18h后向培養(yǎng)瓶中加入溶于二甲基亞砜(DMSO)的阿托伐他汀(終濃度10μM),對照培養(yǎng)瓶中加入等體積的DMSO。培養(yǎng)48h后收集懸浮的細(xì)胞分別標(biāo)記為他汀修飾的DCs (statin-DCs)和未經(jīng)他汀修飾的DCs(untreated-DCs)。通過流式檢測不同組DCs表面CD80、 CD86、 MHC-II分子。 3.動物分組及干預(yù)將EAN大鼠隨機(jī)分為三組,每組五只,在免疫第五天,治療組分別給予他汀修飾的DCs、未經(jīng)他汀修飾的DCs腹腔注射,每只大鼠注射的細(xì)胞數(shù)為1×106個,對照組(control group)給予等體積的1640培養(yǎng)基腹腔注射。 4.制備淋巴結(jié)單個核細(xì)胞(MNC)在EAN發(fā)病高峰期處死大鼠,在無菌條件下取其腹股溝淋巴結(jié),研磨淋巴結(jié),制備MNC并計(jì)數(shù),將細(xì)胞濃度調(diào)整為2×106個細(xì)胞/ml。 5.流式細(xì)胞儀檢測淋巴結(jié)單個核細(xì)胞表面的CD80、 CD86及MHC-Ⅱ取淋巴結(jié)MNC,洗滌后分別加入FITC標(biāo)記的小鼠抗大鼠CD86和MHC-II抗體,以及PE標(biāo)記的小鼠抗大鼠CD80抗體,經(jīng)孵育、重懸、過濾等步驟,流式細(xì)胞儀檢測。 6.流式檢測NK和NKT細(xì)胞取淋巴結(jié)MNC,洗滌后分別加入FITC標(biāo)記的小鼠抗大鼠CD3抗體和PE標(biāo)記的小鼠抗大鼠CD161a抗體,經(jīng)孵育、重懸、過濾等步驟,流式細(xì)胞儀檢測。 7.流式檢測Th1/Th2/Th17型細(xì)胞因子取淋巴結(jié)MNC,經(jīng)洗滌、固定、破膜等步驟,分別加入PE標(biāo)記的小鼠抗大鼠IL-10抗體、FITC標(biāo)記的抗大鼠IFN-γ抗體、PE標(biāo)記的抗大鼠TNF-α抗體和FITC標(biāo)記的抗大鼠IL-17A抗體,經(jīng)孵育、重懸、過濾等步驟,流式細(xì)胞儀檢測。 8.流式檢測Treg細(xì)胞取淋巴結(jié)MNC,洗滌后分別加入PE標(biāo)記的抗CD25抗體(小鼠抗大鼠)和FITC標(biāo)記的抗CD4抗體(小鼠抗大鼠),4℃條件下孵育30min,避光,洗滌后加入固定／破膜工作液并混勻,4℃孵育過夜,避光。洗滌細(xì)胞后加入PE-Cy5標(biāo)記的Foxp3抗體4℃孵育30min,重懸、過濾后,流式細(xì)胞儀檢測。 9.淋巴細(xì)胞增殖實(shí)驗(yàn)將同組細(xì)胞懸液以同樣比例混合,加入0.5μl CFSE,37℃避光孵育30min,加入預(yù)冷的1640,置于冰上孵育5min；離心棄上清后加入完全培養(yǎng)基,以相同細(xì)胞數(shù)種于24孔板,分別加入刺激物BPM或ConA,置于37℃、5%CO2條件下培養(yǎng)72h；將24孔板中的細(xì)胞分別收集到流式管中,離心5min (1200rpm/min),棄上清后混勻；洗滌后加入PE標(biāo)記的抗大鼠CD4抗體,經(jīng)孵育、重懸、過濾等步驟,流式細(xì)胞儀檢測。 10.組織病理學(xué)檢測在發(fā)病高峰期即免疫后第14天,處死大鼠后取坐骨神經(jīng),10%多聚甲醛固定,石蠟包埋,常規(guī)切片,依次經(jīng)脫蠟、水化、蘇木素染色、0.5%伊紅液染色、脫水、透明、封片等步驟,顯微鏡下觀察坐骨神經(jīng)中炎性細(xì)胞的浸潤情況并計(jì)數(shù)。 11.胸腺免疫組化檢測大鼠胸腺石蠟包埋后切片,常規(guī)脫蠟、水化,檸檬酸鹽抗原修復(fù),H202消除內(nèi)源性過氧化物酶,滴加稀釋好的一抗,大鼠抗小鼠/大鼠Foxp3,4℃過夜。復(fù)溫后洗滌,滴加HRP標(biāo)記的羊抗大鼠二抗,37℃孵育1h,洗滌后DAB顯色,蘇木素復(fù)染,脫水、透明、封片。顯微鏡下觀察大鼠胸腺中Foxp3+細(xì)胞的情況并計(jì)數(shù)。 研究結(jié)果： 1.阿托伐他汀對DC表型的影響他汀修飾的DCs較未經(jīng)他汀修飾的DCs表面共刺激分子CD80和CD86的表達(dá)明顯降低,而MHC-II分子沒有顯著性差異。 2.三組大鼠的發(fā)病情況與臨床評分與對照組相比,他汀修飾的DCs在免疫的第11到第14天,明顯緩解了EAN大鼠的臨床癥狀；而與未經(jīng)修飾的DCs相比,他汀修飾的DCs在免疫的第12到第14天,明顯減輕了EAN大鼠的臨床癥狀；對照組與未經(jīng)他汀修飾的DCs治療組的臨床評分之間無明顯差異。 3.阿托伐他汀修飾的DCs治療減少EAN大鼠坐骨神經(jīng)中的炎性細(xì)胞他汀修飾的DCs治療組中EAN大鼠坐骨神經(jīng)中炎性細(xì)胞較未處理DCs治療組及對照組明顯減少。 4.阿托伐他汀修飾的DCs治療下調(diào)EAN大鼠淋巴結(jié)單個核細(xì)胞上CD80、 CD86和MHC-Ⅱ的表達(dá)。與對照組相比,他汀修飾DCs治療組中EAN大鼠淋巴結(jié)單個核細(xì)胞上CD80、 CD86及MHC-II均顯著降低；與未處理DCs治療組相比,上述指標(biāo)亦有降低,但是沒有顯著性差異。而且,淋巴結(jié)單個核細(xì)胞上CD80和CD86在對照組中和未處理DCs治療組中沒有顯著性差異,但是MHC-II在未處理DCs治療組中明顯降低,與對照組相比。 5.他汀修飾的DCs治療降低了EAN大鼠淋巴結(jié)單個核細(xì)胞中Thl和Thl7型細(xì)胞因子的表達(dá)與對照組相比,他汀修飾DCs治療組中EAN大鼠淋巴結(jié)單個核細(xì)胞中的IFN-γ, TNF-α和IL-17A均顯著降低；與未處理DCs治療組相比,IL-17A和TNF-α降低,但無顯著性差異；同時與對照組相比,未處理DCs治療組中IFN-γ和TNF-α顯著下降,而IL-17A在這兩組中無顯著性差異；三組之中,IL-10沒有顯著性差異。 6.他汀修飾的DCs上調(diào)了淋巴結(jié)單個核細(xì)胞中NK和NKT細(xì)胞的數(shù)量與對照組和未處理DCs治療組相比,CD3-CD161a+NK細(xì)胞在他汀修飾DCs治療組明顯升高；與另外兩組相比,CD3+CD161a+NKT細(xì)胞在他汀修飾的DCs治療組也有升高,而且與對照組之間有顯著性差異,而與未處理DCs治療組無顯著性差異。 7.他汀修飾的DCs上調(diào)了淋巴結(jié)單個核細(xì)胞CD4+Foxp3+Treg細(xì)胞的數(shù)量與對照組及未處理DCs治療組相比,他汀修飾的DCs上調(diào)了淋巴結(jié)單個核細(xì)胞中CD4+Foxp3+Treg細(xì)胞的數(shù)量；而在其他兩組之間未發(fā)現(xiàn)CD4+Foxp3+Treg細(xì)胞數(shù)量有顯著性差異。 8.他汀修飾DCs上調(diào)了胸腺中Foxp3+細(xì)胞的數(shù)量與對照組及未處理DCs治療組相比,他汀修飾DCs治療組中胸腺Foxp3+細(xì)胞的數(shù)量明顯增加；另外兩組中Foxp3+細(xì)胞數(shù)量無顯著性差異。 9.他汀修飾的DCs治療抑制了淋巴細(xì)胞增殖反應(yīng)與對照組及未處理DCs組相比,他汀修飾DCs顯著抑制了淋巴細(xì)胞增殖反應(yīng),無論是在BPM抗原刺激的情況下,還是ConA抗原刺激的情況下；我們沒有發(fā)現(xiàn)在對照組和未處理DCs治療組之間,淋巴細(xì)胞增殖有顯著性差異。 結(jié)論： 1.阿托伐他汀可誘導(dǎo)DCs成為耐受性DCs。 2.阿托伐他汀修飾的DCs干預(yù),可以減輕實(shí)驗(yàn)性自身免疫性神經(jīng)炎的癥狀。 3.阿托伐他汀修飾的DCs在實(shí)驗(yàn)性自身免疫性神經(jīng)炎中可誘導(dǎo)中樞(胸腺)和外周(淋巴結(jié))免疫耐受。 意義： 本研究通過阿托伐他汀誘導(dǎo)DCs成為耐受性DCs,給予EAN大鼠腹腔注射,顯示他汀誘導(dǎo)的耐受性DCs能夠減輕EAN的癥狀并誘導(dǎo)免疫耐受,這種作用主要是通過抑制淋巴結(jié)單個核細(xì)胞表面共刺激分子(CD80和CD86)及MHC-Ⅱ的表達(dá)、降低Thl/Th17型細(xì)胞因子、上調(diào)淋巴結(jié)中Treg細(xì)胞及胸腺中Foxp3細(xì)胞的表達(dá)及上調(diào)淋巴結(jié)單個核細(xì)胞中NK及NKT細(xì)胞數(shù)量實(shí)現(xiàn)的。本研究為人類GBS的治療提供了新的思路。
[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錛，

本文編號：1830625