马索罗酚和丹参酮ⅡA对实验性自身免疫性脑脊髓炎小鼠脾淋巴细胞的抗氧化应激和免疫调节作用

发布时间：2018-05-18 11:35

  本文选题：多发性硬化 + 实验性自身免疫性脑脊髓炎　； 参考：《河北医科大学》2014年硕士论文

【摘要】：目的：多发性硬化（Multiple Sclerosis, MS），是累及中枢神经系统（CNS）的一种慢性炎症性脱髓鞘性疾病，发病机制尚不明确。大多数研究表明，免疫损伤和氧化应激在MS的发病机制中起重要作用。Nrf2/ARE通路的激活能够保护机体免受免疫及氧化应激的损伤，本课题组前期应用Nrf2通路的经典激活剂莱菔硫烷初步证实了激活此通路能够降低EAE发病率以及减轻疾病严重程度。而莱菔硫烷尚未用于临床，我们希望通过实验，从已经用于临床的Nrf2通路激活性药物中筛选出抗氧化、调节免疫等方面可以跟莱菔硫烷媲美的药物，从而为MS的治疗提出新的药物。马索罗酚是一种化疗药物，丹参酮IIA常用于冠心病的治疗，此两者都被证实具有Nrf2通路激活剂的作用，本实验利用实验性自身免疫性脑脊髓炎（ExperimentalAutoimmune Encephalomyelitis, EAE）小鼠的脾细胞，研究马索罗酚和丹参酮IIA对EAE小鼠脾细胞中抗氧化酶、细胞因子IFN-γ、IL-4、IL-10、IL-17A的表达的调节作用，初步探讨马索罗酚和丹参酮IIA对EAE的抗氧化和免疫调节作用，一方面希望为MS的治疗提出新的药物，另一方面希望能挖掘这两种药物在调节免疫方面的特性。 方法：选择8只C57BL/6雌性小鼠，，周龄8-10周，体重18-22g,利用MOG35-55多肽、结核杆菌、完全弗氏佐剂、百日咳毒素等对小鼠进行免疫制作成EAE模型，每天2次对其进行神经功能的评分，于发病高峰（8分及以上，约免疫后20天）随机选择5只处死，在无菌的环境中取出脾脏，并用10%胎牛血清的RPMI1640无菌培液制备成单细胞悬液（细胞密度控制在5*106/L左右），并给予MOG35-55多肽进行再刺激。将此细胞悬液随机分为对照组、莱菔硫烷组、马索罗酚组和丹参酮IIA组，空白对照组加入无菌DMSO（0.6l/200ul）莱菔硫烷组加入莱菔硫烷稀释液（0.9g/ml）马索罗酚组加入马索罗酚稀释液（6.0g/ml）丹参酮IIA组加入丹参酮IIA稀释液（3.0g/ml），保证所有组的DMSO含量均≤0.3%。将4组细胞悬液培养24小时后离心，利用Western blot的方法检测细胞沉渣中的Ⅰ型血红素氧化酶HO-1，采用ELISA的方法检测上清液中的IFN-γ、IL-4、IL-10、IL-17A的表达水平。 结果： 1小鼠脾细胞中HO-1表达情况：莱菔硫烷组、马索罗酚组、丹参酮IIA组EAE小鼠脾细胞与对照组相比，HO-1的表达明显增多，差异均具有统计学意义（P0.05）。丹参酮IIA组HO-1表达明显高于莱菔硫烷组及马索罗酚组（P0.05）；莱菔硫烷组与马索罗酚组HO-1表达相比，差异无统计学意义（P＞0.05）。 2上清液中IFN-γ、IL-4、IL-10、IL-17A的表达情况：莱菔硫烷组、马索罗酚组、丹参酮IIA组与对照组相比，IFN-γ的表达无明显统计学差异（P＞0.05）；IL-4、IL-10的表达明显增多，差异均具有统计学意义（P0.05）；IL-17A的表达明显减少，差异均具有统计学意义（P0.05）；莱菔硫烷组、马索罗酚组及丹参酮IIA组IL-4、IL-10、IL-17A的表达相比较，差异无统计学意义（P＞0.05）。 结论： 1马索罗酚、丹参酮IIA都能够上调EAE小鼠脾淋巴细胞中的IL-4、IL-10等抑炎因子的表达，下调促炎因子IL-17A的表达，具有调节免疫平衡的能力。 2马索罗酚、丹参酮IIA能够上调EAE小鼠脾淋巴细胞中的HO-1的表达，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.
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