本文關(guān)鍵詞：負(fù)性協(xié)同刺激分子PD-L1在實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎中的表達(dá)　出處：《蘇州大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 多發(fā)性硬化 實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎 協(xié)同刺激分子 PD-L1分子

【摘要】： 背景與目的 多發(fā)性硬化(multiple sclerosis, MS)是CD4+Th1細(xì)胞介導(dǎo)的中樞神經(jīng)系統(tǒng)(central nervous system, CNS)脫髓鞘為主的自身免疫性疾病。協(xié)同刺激分子PD-Ll分子是新近發(fā)現(xiàn)的B7家族的負(fù)性協(xié)同刺激分子之一,該分子通過(guò)與主要表達(dá)在活化的T細(xì)胞、B細(xì)胞的抑制性受體PD-l分子結(jié)合發(fā)揮負(fù)性免疫調(diào)節(jié)作用。實(shí)驗(yàn)性變態(tài)反應(yīng)性腦脊髓炎(experimental allergic encephalomyelitis,EAE)的病理變化和發(fā)病機(jī)制與MS極其相似,是研究人類(lèi)多發(fā)性硬化的理想動(dòng)物模型。 為了進(jìn)一步分析PD-L1分子在EAE和MS中可能的免疫作用,本實(shí)驗(yàn)用周齡和性別匹配的C57BL/6J小鼠成功建立了EAE動(dòng)物模型,并檢測(cè)負(fù)性協(xié)同刺激分子PD-L1在EAE中的表達(dá),以便進(jìn)一步探討PD-L1/PD-1途徑在發(fā)病機(jī)制中的作用,為人類(lèi)MS的臨床治療提供可能的思路。 方法 第一部分:30只雌性C57BL/6J小鼠隨機(jī)分為兩組,實(shí)驗(yàn)組20只和對(duì)照組10只。實(shí)驗(yàn)組用髓鞘少突膠質(zhì)細(xì)胞糖蛋白(myelin oligodendrocyte glycoprotein, MOG35-55)加完全弗氏佐劑(complete freund adjuvant,CFA)制備抗原佐劑乳化物腋下皮下單點(diǎn)單次注射免疫小鼠,分別在免疫當(dāng)天和24小時(shí)后尾靜脈注射百日咳毒素(bordetella pertussis vaccine,BPV),對(duì)照組皮下和尾靜脈注射PBS,觀察兩組動(dòng)物的發(fā)病臨床表現(xiàn)并按照國(guó)際通用的五分制評(píng)分法進(jìn)行癥狀分級(jí)評(píng)分,于免疫后12-14天取對(duì)照組和癥狀明顯的實(shí)驗(yàn)組小鼠的脊髓進(jìn)行冰凍切片并行HE染色、Luxol Fast-Blue髓鞘染色以鑒定模型成功與否。 第二部分:免疫組化檢測(cè)脊髓組織PD-L1的表達(dá),并用流式細(xì)胞儀(Flowcytometry,FCM)、Weston-Blot方法檢測(cè)兩組動(dòng)物的脾細(xì)胞上PD-L1的表達(dá)。 結(jié)果 1.實(shí)驗(yàn)組小鼠最早于抗原誘導(dǎo)后的第10天左右開(kāi)始出現(xiàn)動(dòng)作遲緩、尾部無(wú)力等癥狀,以后逐漸加重,于免疫后12-14天癥狀達(dá)高峰,有的甚至大小便失禁、死亡,16-19天癥狀開(kāi)始緩解,但癥狀不能完全緩解,對(duì)照組小鼠則無(wú)任何臨床癥狀,共觀察小鼠30天。 2.切片HE染色光鏡下見(jiàn)實(shí)驗(yàn)組小鼠脊髓組織血管周?chē)罅垦仔约?xì)胞浸潤(rùn),呈“袖套”樣改變,對(duì)照組小鼠未見(jiàn)明顯病理改變;LFB髓鞘染色光鏡下見(jiàn)實(shí)驗(yàn)組急性期小鼠脊髓組織的白質(zhì)髓鞘脫失明顯,對(duì)照組小鼠無(wú)此改變。 3.免疫組化顯示實(shí)驗(yàn)組小鼠脊髓組織PD-L1分子的表達(dá)明顯增加,流式細(xì)胞儀(Flow cytometry FCM)、Weston-Blot檢測(cè)實(shí)驗(yàn)組動(dòng)物的脾細(xì)胞上PD-L1分子的表達(dá)明顯增加。 結(jié)論 1.本實(shí)驗(yàn)建立的小鼠模型在臨床表現(xiàn)、病理變化等方面符合復(fù)發(fā)-非緩解型EAE的表現(xiàn),模型穩(wěn)定、可靠,是研究MS的理想動(dòng)物模型。 2.實(shí)驗(yàn)組動(dòng)物的PD-L1的表達(dá)在外周脾細(xì)胞和中樞神經(jīng)系統(tǒng)的脊髓組織都較對(duì)照組明顯增加。
[Abstract]:Background and purpose Multiple sclerosis multiple sclerosis. S) is a central nervous system mediated by CD4 Th1 cells. Costimulator PD-Ll is one of the newly discovered negative co-stimulators of B7 family. The molecule is expressed mainly in activated T cells. The inhibitory receptor PD-l molecules of B cells play a negative immunoregulatory role. Experimental allergic Encephalomyelitis. Experimental allergic encephalomyelitis. EAE is an ideal animal model for the study of multiple sclerosis. In order to further analyze the possible immunological effects of PD-L1 molecules in EAE and MS, EAE animal models were successfully established in C57BL / 6J mice with week-old and sex-matched C57BL / 6J mice. The expression of PD-L1 in EAE was detected in order to further explore the role of PD-L1/PD-1 pathway in pathogenesis. To provide possible ideas for clinical treatment of human MS. Method Part one: 30 female C57BL / 6J mice were randomly divided into two groups. The experimental group (n = 20) and the control group (n = 10) were treated with myelin oligodendrocyte glycoprotein. MOG35-55) complete freund adjuvant with complete Freund's adjuvant. CFA) was used to prepare antigenic adjuvant emulsifiers for subaxillary subcutaneous single injection to immunize mice. Pertussis toxin Bordetella pertussis vas was injected intravenously on the day and 24 hours after immunization. PBSs were injected subcutaneously and caudally in the control group. The clinical symptoms of the two groups were observed and the symptoms were graded according to the international common five-point scoring method. At 12-14 days after immunization, the spinal cord of the control group and the experimental group with obvious symptoms were taken for frozen sections and HE staining. Luxol Fast-Blue myelin staining was used to identify the success of the model. The second part: immunohistochemistry was used to detect the expression of PD-L1 in spinal cord and flow cytometry was used to detect the expression of FCM. Weston-Blot method was used to detect the expression of PD-L1 on spleen cells of both groups. Results 1. The mice in the experimental group began to appear the symptoms of slow movement and tail weakness at the first 10 days after antigen induction, then gradually aggravated, and reached the peak at 12-14 days after immunization. Some even incontinence, the death of 16-19 days symptoms began to relieve, but the symptoms can not be completely relieved, the control mice without any clinical symptoms, a total of 30 days observation of mice. 2. Under HE staining, a large number of inflammatory cells were infiltrated around the blood vessels of the spinal cord in the experimental group, but no obvious pathological changes were observed in the control group. LFB staining showed that the myelin demyelination of the white matter in the acute phase of the experimental group was obvious, but that in the control group was not. 3.Immunohistochemistry showed that the expression of PD-L1 in the spinal cord of the experimental group was significantly increased, flow cytometry flow cytometry (FCM). The expression of PD-L1 in spleen cells of experimental group was detected by Weston-Blot. Conclusion 1. The mouse model established in this study is stable and reliable in clinical manifestations and pathological changes. It is an ideal animal model for the study of MS. 2. The expression of PD-L1 in peripheral spleen cells and spinal cord of central nervous system in experimental group was significantly higher than that in control group.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392

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