本文選題：多發(fā)性硬化 + 實(shí)驗(yàn)性自身免疫性腦脊髓炎��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：多發(fā)性硬化（Multiple Sclerosis, MS），是累及中樞神經(jīng)系統(tǒng)（CNS）的一種慢性炎癥性脫髓鞘性疾病，發(fā)病機(jī)制尚不明確。大多數(shù)研究表明，免疫損傷和氧化應(yīng)激在MS的發(fā)病機(jī)制中起重要作用。Nrf2/ARE通路的激活能夠保護(hù)機(jī)體免受免疫及氧化應(yīng)激的損傷，本課題組前期應(yīng)用Nrf2通路的經(jīng)典激活劑萊菔硫烷初步證實(shí)了激活此通路能夠降低EAE發(fā)病率以及減輕疾病嚴(yán)重程度。而萊菔硫烷尚未用于臨床，我們希望通過(guò)實(shí)驗(yàn)，從已經(jīng)用于臨床的Nrf2通路激活性藥物中篩選出抗氧化、調(diào)節(jié)免疫等方面可以跟萊菔硫烷媲美的藥物，從而為MS的治療提出新的藥物。馬索羅酚是一種化療藥物，丹參酮IIA常用于冠心病的治療，此兩者都被證實(shí)具有Nrf2通路激活劑的作用，本實(shí)驗(yàn)利用實(shí)驗(yàn)性自身免疫性腦脊髓炎（ExperimentalAutoimmune Encephalomyelitis, EAE）小鼠的脾細(xì)胞，研究馬索羅酚和丹參酮IIA對(duì)EAE小鼠脾細(xì)胞中抗氧化酶、細(xì)胞因子IFN-γ、IL-4、IL-10、IL-17A的表達(dá)的調(diào)節(jié)作用，初步探討馬索羅酚和丹參酮IIA對(duì)EAE的抗氧化和免疫調(diào)節(jié)作用，一方面希望為MS的治療提出新的藥物，另一方面希望能挖掘這兩種藥物在調(diào)節(jié)免疫方面的特性。 方法：選擇8只C57BL/6雌性小鼠，，周齡8-10周，體重18-22g,利用MOG35-55多肽、結(jié)核桿菌、完全弗氏佐劑、百日咳毒素等對(duì)小鼠進(jìn)行免疫制作成EAE模型，每天2次對(duì)其進(jìn)行神經(jīng)功能的評(píng)分，于發(fā)病高峰（8分及以上，約免疫后20天）隨機(jī)選擇5只處死，在無(wú)菌的環(huán)境中取出脾臟，并用10%胎牛血清的RPMI1640無(wú)菌培液制備成單細(xì)胞懸液（細(xì)胞密度控制在5*106/L左右），并給予MOG35-55多肽進(jìn)行再刺激。將此細(xì)胞懸液隨機(jī)分為對(duì)照組、萊菔硫烷組、馬索羅酚組和丹參酮IIA組，空白對(duì)照組加入無(wú)菌DMSO（0.6l/200ul）萊菔硫烷組加入萊菔硫烷稀釋液（0.9g/ml）馬索羅酚組加入馬索羅酚稀釋液（6.0g/ml）丹參酮IIA組加入丹參酮IIA稀釋液（3.0g/ml），保證所有組的DMSO含量均≤0.3%。將4組細(xì)胞懸液培養(yǎng)24小時(shí)后離心，利用Western blot的方法檢測(cè)細(xì)胞沉渣中的Ⅰ型血紅素氧化酶HO-1，采用ELISA的方法檢測(cè)上清液中的IFN-γ、IL-4、IL-10、IL-17A的表達(dá)水平。 結(jié)果： 1小鼠脾細(xì)胞中HO-1表達(dá)情況：萊菔硫烷組、馬索羅酚組、丹參酮IIA組EAE小鼠脾細(xì)胞與對(duì)照組相比，HO-1的表達(dá)明顯增多，差異均具有統(tǒng)計(jì)學(xué)意義（P0.05）。丹參酮IIA組HO-1表達(dá)明顯高于萊菔硫烷組及馬索羅酚組（P0.05）；萊菔硫烷組與馬索羅酚組HO-1表達(dá)相比，差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 2上清液中IFN-γ、IL-4、IL-10、IL-17A的表達(dá)情況：萊菔硫烷組、馬索羅酚組、丹參酮IIA組與對(duì)照組相比，IFN-γ的表達(dá)無(wú)明顯統(tǒng)計(jì)學(xué)差異（P＞0.05）；IL-4、IL-10的表達(dá)明顯增多，差異均具有統(tǒng)計(jì)學(xué)意義（P0.05）；IL-17A的表達(dá)明顯減少，差異均具有統(tǒng)計(jì)學(xué)意義（P0.05）；萊菔硫烷組、馬索羅酚組及丹參酮IIA組IL-4、IL-10、IL-17A的表達(dá)相比較，差異無(wú)統(tǒng)計(jì)學(xué)意義（P＞0.05）。 結(jié)論： 1馬索羅酚、丹參酮IIA都能夠上調(diào)EAE小鼠脾淋巴細(xì)胞中的IL-4、IL-10等抑炎因子的表達(dá)，下調(diào)促炎因子IL-17A的表達(dá)，具有調(diào)節(jié)免疫平衡的能力。 2馬索羅酚、丹參酮IIA能夠上調(diào)EAE小鼠脾淋巴細(xì)胞中的HO-1的表達(dá)，具有抗氧化的能力。
[Abstract]:Objective: Multiple Sclerosis (MS) is a chronic inflammatory demyelinating disease involving the central nervous system (CNS). The pathogenesis is not clear. Most studies have shown that immune injury and oxidative stress play an important role in the pathogenesis of MS, and the activation of.Nrf2/ARE pathway protects the body from immunity and oxidation. Stress damage, the classic activator of Nrf2 pathway in our group has preliminarily confirmed that the activation of this pathway can reduce the incidence of EAE and reduce the severity of the disease. However, sulforaphane has not been used in clinical trials. We hope to screen out antioxidative agents from the Nrf2 pathway activators that have been used in clinical trials. A new drug that can be compared with sulforaphane, which can be compared with sulforaphane, is a new drug for the treatment of MS. It is a chemotherapeutic drug. Tanshinone IIA is commonly used in the treatment of coronary heart disease. Both are proved to have the role of Nrf2 pathway activator. This experiment uses experimental autoimmune encephalomyelitis (ExperimentalAutoimmune). Encephalomyelitis, EAE) mice spleen cells, study the regulation of the expression of anti oxidase, cytokine IFN- gamma, IL-4, IL-10, IL-17A in the spleen cells of EAE mice, and preliminarily discuss the antioxidant and immunomodulatory effects of salviolol and tanshinone IIA on EAE in EAE mice. On the one hand, we hope to provide a new drug for MS treatment. On the other hand, we hope to explore the characteristics of these two drugs in regulating immunity.
Methods: 8 C57BL/6 female mice were selected for 8-10 weeks of age and weight of 18-22g. The mice were immunized with MOG35-55 peptide, Mycobacterium tuberculosis, complete Freund's adjuvant and pertussis toxin to make EAE model. The neurological function was evaluated 2 times a day, and 5 died randomly at the peak of onset (8 points and above, about 20 days after immunization). The spleen was removed and the single cell suspension was prepared by the RPMI1640 aseptic culture of the 10% fetal bovine serum (the cell density was controlled at about 5*106/L), and the MOG35-55 polypeptide was re stimulated. The suspension was randomly divided into the control group, the sulforaphane group, the carrophenol group and the tanshinone IIA group, and the blank control group was added to the aseptic DMSO (0.6l/). 200ul) sulforaphane group added to the sulforaphane diluent (0.9g/ml) group to add the IIA group of tanshinone (6.0g/ml) into the tanshinone IIA diluent (3.0g/ml), and ensure that the DMSO content of all groups is less than 0.3%. and the 4 groups of cell suspension are isolated from the heart for 24 hours, and the type I in the cell sediment is detected by the Western blot method. Heme oxygenase HO-1 was used to detect the expression levels of IFN-, IL-4, IL-10 and IL-17A in the supernatant by ELISA.
Result錛

本文編號(hào)：1905681