1、12022/8/712022/8/7免疫系統(tǒng)狼瘡/硬皮病 多發(fā)性硬化重癥肌無力類風濕性關(guān)節(jié)炎潰瘍性腸炎格林巴利 22022/8/722022/8/7 Experimental Autoimmune Encephalomyelitis an animal model for Multiple SclerosisDepartment of Neurobiology32022/8/7以中樞神經(jīng)系統(tǒng)白質(zhì)脫髓鞘病變?yōu)樘卣鞯淖陨砻庖咝约膊?。病灶部位和時間上的多發(fā)性為其臨床特點多發(fā)性硬化(Multiple sclerosis, MS)（一）. 概述42022/8/7神經(jīng)纖維：有髓和無髓髓鞘：包裹某些神經(jīng)軸突

2、的蛋白質(zhì)和脂質(zhì)組成的膜狀結(jié)構(gòu)。 周圍神經(jīng)-Schwann cell（一對一) 中樞神經(jīng)-Oligodendrocyte（一對多）髓鞘的作用：絕緣、保護軸突，有利神經(jīng)沖動的傳導52022/8/7 脫髓鞘疾?。?以髓鞘脫失為共同病理特征，但病因不同、臨床表現(xiàn)各異的疾病的統(tǒng)稱。62022/8/762022/8/772022/8/772022/8/7無髓神經(jīng)纖維上是以局部電流的形式進行傳導有髓神經(jīng)纖維外面包裹著一層電阻很高的髓鞘，動作電位只能在沒有髓鞘的朗飛氏結(jié)處產(chǎn)生局部電流82022/8/782022/8/792022/8/7以中樞神經(jīng)系統(tǒng)白質(zhì)脫髓鞘病變?yōu)樘卣鞯淖陨砻庖咝约膊?。病灶部位和時間上的多發(fā)

3、性為其臨床特點多發(fā)性硬化(Multiple sclerosis,MS)（一）. 概述102022/8/7102022/8/71、臨床體征112022/8/72、MS臨床分型：倫敦MS自然病史人群（1972-1984起始發(fā)?。?043位MS824位發(fā)作起病219位進展起病273位復發(fā)緩解型71位單次發(fā)作進展型480位復發(fā)進展型61位進展復發(fā)型158位原發(fā)進展型132022/8/7炎癥正常膠質(zhì)增生脫髓鞘3、病理特點142022/8/7隨緯度的增加患病率有增高的趨勢 北歐和北美國家年發(fā)病率為30-80/100000，赤道 地區(qū)年發(fā)病率為1/100000 我國和其他亞洲國家一樣，是該病的低發(fā)區(qū) 4、病

4、因環(huán)境因素（特殊地理分布）152022/8/7152022/8/7環(huán)境因素（特殊地理分布）Sodium chloride drives autoimmune disease by the induction of pathogenic Th17 cells Department of Neurology and ImmunobiologyYale School of MedicineNew Haven，USADavid A. Hafler 172022/8/7遺傳因素約15%具家族史，單卵雙生發(fā)病概率高達34%。發(fā)病率與HLA（human leucocyte antigen）有關(guān)，特別是HLA

5、-DR2，另外尚有HLA-DR3、HLA-B7、HLA-A3等MS病人親屬發(fā)病風險率同MS病人的關(guān)系 MS的發(fā)病風險一級親屬 3.0%女性單合子孿生兄妹 34%女性雙合子孿生兄妹 5.4%被收養(yǎng)的子女 0.2%同父異母兄妹 1.2%同母異父兄妹 2.2%婚配的子女 30.5%30歲以下發(fā)病的男性MS病人的姐妹 12.7%192022/8/7病毒感染學說乙肝病毒、HHV-6（h u m a n p a p i l l o m a v i r u s） 、EBV (Epstein-Barr virus)并未找到抗原等病毒直接感染的依據(jù)自身免疫學說202022/8/7Immunopathogenes

6、is of multiple sclerosis.5、發(fā)病機制212022/8/7 The ideograph of Th cell activation during EAE proceeding232022/8/7T cell classification232022/8/7MSC262022/8/7262022/8/7Th17MS272022/8/7272022/8/7Fig. (a). IL-17 staining was observed in multiple sclerosis lesions, but not in control CNS material.Fig. (b).

7、IL-17R staining was observed on endothelial cells in multiple sclerosis lesions, but not in controls.abSodium chloride drives autoimmune disease by the induction of pathogenic Th17 cells Department of Neurology and ImmunobiologyYale School of MedicineNew Haven，USADavid A. Hafler High-salt diet induc

8、es Th17 cells in vivo and exacerbates experimental autoimmune encephalomyelitis. to examine the effects of increased NaCl in an in vivo system.Brain leukocytes were isolated on day 17 p.i. of EAE and analysed by FACS for IL-17A and CD4. Cytokine concentrations in supernatants. Rats were sacrificed 7

9、, 14 and 21 d and purified lymphocytes were stimulated. The cytokine concentrations were measured by ELISA. CD4+ T cell profiles in EAE rats. Lymphocytes from four groups were isolated on day after immunization. CD4+ cell expression of IFN-, IL-4, IL-17 and Foxp3 was assessed by flow cytometry. 3320

10、22/8/7332022/8/7Blood Brain Barrier，BBB （血腦屏障）保羅埃爾利希352022/8/7352022/8/7362022/8/7362022/8/7372022/8/7372022/8/7Fig. (A) Western blot for the tight-junction proteins occludin, ZO-1 and junction adhesion molecule (JAM)-1 from human BBB-ECs Fig. (B) Western blot for tight-junction proteins in spinal c

11、ord homogenates of EAE mice. In situ immunostaining for ZO-1 (red) and nuclear stain TO-PRO3 (blue)ABA為TRITC標記的GFAP，呈紅色；B為TRITC與DAPI標記細胞核的合成圖（x200） ；星形膠質(zhì)細胞純度約91%ABProliferation of MOG35-55 specific lymphocytes cocultured with astrocytes at varies lymphocyte/astrocyte ratios.* P 0.05, * P 0.01, * P 0

12、.001Cytokine levels in lymphocytes culture supernatants. * P 0.001IL-27 production by astrocytes after cocultured with lymphocytes.* P 0.05Cytokine levels in lymphocytes culture supernatants after IL-27 neutrilization.*P 0.01, *P 0.001IFN-誘導的星形膠質(zhì)細胞MHC-的表達Inducible MHC- expression on astrocytes in vi

13、tro.* P 0.001AEAE發(fā)病中脊髓MHC-的表達 MHC- expression in the spinal cords was coincided with EAE course. * P 0.05A EAE發(fā)病中脊髓星形膠質(zhì)細胞上MHC-的表達A,D,G為TRITC標記的GFAP; B,E,H為FITC標記的MHC-; C,F,I為合成圖 (200)。箭頭所指為雙陽性細胞，Scale bar = 50 m. Expression of MHC- on astrocytes during EAE. ELISA檢測培養(yǎng)上清中細胞因子含量淋巴細胞與激活的星形膠質(zhì)細胞培養(yǎng)上清 Cytok

14、ine levels in lymphocytes culture supernatants. * P 0.001 RT-PCR法檢測星形膠質(zhì)細胞MHC-表達 MHC- mRNA levels in astrocytes after cocultured with lymphocytes.* P 0.01, * P 0.001實驗結(jié)論1 在EAE發(fā)病過程中，星形膠質(zhì)細胞發(fā)揮著促進和抑制致病性淋巴細胞的雙重作用；2 在EAE疾病起始階段，星形膠質(zhì)細胞通過分泌IL-27抑制浸潤的淋巴細胞增殖及分化；3 在EAE疾病效應階段，星形膠質(zhì)細胞在IFN-作用下轉(zhuǎn)化為抗原提呈細胞，促進致病性淋巴細胞作用，從

15、而加速疾病的進展。492022/8/7492022/8/71933-35年 在猴子身上反復注射正常兔子的腦組織成分，長達半年甚至1年半的觀察發(fā)現(xiàn)的，后來相繼在兔子和豬等動物中誘發(fā)出相應的模型。 (二). 實驗性自身免疫性腦脊髓炎 (Experimental autoimmune encephalomyelitis, EAE)502022/8/7502022/8/7Olitsky, P.K. and Yager, R.H. 1949. Experimental disseminated ncephalomyelitis in white mice. J. Exp. Med. 90:213-223

16、.Mouse model including: acute; chronic or replase EAE512022/8/7512022/8/7EAE模型的建立方法: 主動免疫 (Active immunization) 被動免疫 (Passive imunization or Adoptive transfer)522022/8/7522022/8/7實驗動物髓鞘蛋白完全弗式佐劑百日咳毒素1.實驗材料532022/8/7532022/8/72）髓鞘蛋白PLP（Proteolipid protein）：蛋白脂質(zhì)蛋白MOG(Myelin oligodendrocyte glycoprotein

17、)：髓鞘少突膠質(zhì)細胞糖蛋白MBP（Myelin basic protein）：髓磷脂堿性蛋白注意：其他小鼠品系中主動或者過繼誘導EAE也許需要對方法進行修改以確保能夠有效地引發(fā)動物模型，應該詳細查閱相關(guān)文獻542022/8/7542022/8/7552022/8/7552022/8/7不完全佛式佐劑（ plete Freunds adjuvant，IFA ）結(jié)核菌素（Mycobacterium tuberculosis H37Ra）3）完全佛式佐劑562022/8/7562022/8/7pertussis toxin，PT（200-300 ng/time for twice） 13ml聚苯乙烯

18、試管18 and 25G針頭小動物除毛器4）百日咳毒素及其他572022/8/7572022/8/7IFATBPT582022/8/72022/8/72.抗原準備抗原乳化592022/8/73.皮下免疫602022/8/7602022/8/7評定標準0:No deficit0.5:Partial loss of tail tone or slightly abnormal gait;1.0:Complete tail paralysis or both partial loss of tail tone and mild hind limb weakness;1.5:Complete tail

19、 paralysis and mild hind limb weakness; 2.0:Tail paralysis with moderate hind limb weakness (evidenced by frequent foot dropping between bars of cage top while walking);2.5:No weight-bearing on hind limbs (dragging) but with some legmovement;3.0:Complete hind limb paralysis with no residual movement

20、; 3.5:Hind limb paralysis with mild weakness in forelimbs; 4.0:Complete quadriplegia but with some movement of head; 4.5:Moribund; 5.0:Dead.4.臨床評分和體重監(jiān)測612022/8/7EAE clinical scores612022/8/7622022/8/7622022/8/7632022/8/7642022/8/7ABDC圖3.脊髓組織HE染色及LFB染色。A：CFA組HE染色 B：EAE組HE染色，箭頭所示為炎性細胞浸潤 C：CFA組LFB染色 D：

21、EAE組LFB染色，如箭頭為脫髓鞘斑塊；圖A，B，C，D中左側(cè)放大倍數(shù)，40；右側(cè)放大倍數(shù)，200。652022/8/7652022/8/7注意事項1、實驗動物的選擇與抗原的選擇需對應2、抗原的乳化3、百日咳毒素的注射方式及劑量4、局部免疫的回流5、動物的飼養(yǎng)和觀察662022/8/7672022/8/7672022/8/7Adoptive transfer EAEAT-EAE: 疾病效應階段模型通過預先激活的一群髓鞘表位特異性CD4+T細胞通過外周轉(zhuǎn)移到正常小鼠體內(nèi)Myelin-specific CD4+ T682022/8/7692022/8/7702022/8/7Th1 cells in

22、itiate EAE upon passive transfer and promote therecruitment of Th17 cells to the CNS712022/8/7712022/8/7注意事項1、實驗動物的準備2、淋巴細胞的活性3、百日咳毒素的注射4、動物的飼養(yǎng)和觀察722022/8/7722022/8/7EAE的作用EAE作為研究臨床復發(fā)-緩解型臨床疾病的模型慢性疾病過程中T細胞反應的進展治療中自身免疫性疾病不同免疫調(diào)節(jié)策略的有效性732022/8/7732022/8/7 EAE是一種CD4+T細胞介導的自身免疫性疾病 特征CNS血管周圍CD4+T細胞和單核細胞浸潤，

23、繼發(fā)軸突原發(fā)性脫髓鞘，導致進行后肢癱瘓 EAE提供了一種模型研究CD4+Th1/Th17細胞介導的組織損傷病理過程和免疫調(diào)節(jié)，是公認的人類多發(fā)性硬化相關(guān)模型742022/8/7742022/8/7EAE in rats雖然大鼠特異性的細胞因子和單克隆抗體相對缺乏，但是大鼠已經(jīng)被證明在研究EAE的過程中非常有用 用低劑量的MBP和弗氏佐劑免疫時,能誘發(fā)大鼠發(fā)生嚴重的癱瘓性EAE，表明這種大鼠是一種敏感的亞型 相對于大多數(shù)小鼠的EAE方法，需要用輔助佐劑（比如百日咳毒素，在小鼠中需要用來引發(fā)EAE） 在大鼠中是不需要的在Lewis近交系大鼠中，過繼轉(zhuǎn)移實驗和免疫遺傳學很容易進行 752022/8/

24、7752022/8/7骨化三醇治療實驗性自身免疫性腦脊髓炎的實驗研究 實驗設(shè)計體內(nèi)實驗EAE組CFA組HE染色淋巴細胞免疫后第7天、15天與第21天取材快藍染色FACs檢測 脊髓（腰骶段）治療組C57BL/6雌鼠體外實驗免疫后第7天、15天與第21天提取EAE小鼠淋巴細胞 0處理組（EAE組）淋巴細胞增殖實驗FACs檢測體外藥物干預組C57BL/6雌鼠淋巴細胞(一)、成功建立小鼠EAE模型1.CFA小鼠與EAE小鼠臨床表現(xiàn)圖1.CFA組和EAE組小鼠臨床表現(xiàn)。A: CFA組小鼠，B: EAE組小鼠. AB2.CFA組與EAE組臨床評分 圖2.CFA組和EAE組臨床評分EAE組與CFA組比較，*

25、P0.05， *P0.01， *P0.001.3. CFA組與EAE組小鼠脊髓HE染色結(jié)果圖3.脊髓組織HE染色A: CFA組40， B: EAE組40，C: CFA組200，D: EAE組200.ACBD4.CFA組與EAE組小鼠脊髓快藍染色結(jié)果BDAC圖4.脊髓組織快藍染色.A: CFA組40， B: EAE組40，C: CFA組200，D: EAE組200.(二)、體內(nèi)治療實驗1.臨床評分圖5.骨化三醇治療組和EAE組臨床評分骨化三醇治療組與EAE組相比較，*P0.05，*P0.01，*P0.001.2.CFA、EAE和治療組脊髓HE染色結(jié)果（200）15天7天 CFA組 EAE組 治療組21天圖6.CFA組、EAE組和治療組脊髓HE染色結(jié)果（200）箭頭所示為炎細胞浸潤21天15天 CFA組 EAE組 治療組7天3.CFA、EAE和治療組脊髓快