MAPKs对NOD小鼠IL-12表达及C57小鼠巨噬细胞发育的影响

发布时间：2018-06-18 15:53

  本文选题：MAPKs + 小鼠　； 参考：《河南大学》2008年硕士论文

【摘要】： 背景 丝裂原活化蛋白激酶(MAPKs)系统是进化中最古老最保守的信号转导系统之一,在免疫应答过程中起重要作用。在哺乳动物细胞中主要有三个亚家族,分别为细胞外信号调节激酶(ERK1/2)、c-Jun N端激酶(JNK)/应激活化蛋白激酶(SAPK)、和p38蛋白激酶。MAPKs通过三肽基序苏氨酸-Xaa-酪氨酸发生双磷酸化而活化。该三肽基序序列在每个MAPKs中是不同的:ERK (苏氨酸-谷氨酸-酪氨酸);JNK(苏氨酸-脯氨酸-酪氨酸);和p38(苏氨酸-甘氨酸-酪氨酸)。苏氨酸和酪氨酸的双磷酸化被保守的蛋白激酶系统调控。ERK MAPK被MAPK激酶(MKK) MEK 1和MEK 2激活;JNK MAPK被MKK 4和MKK 7激活;p38 MAPK通过MKK 3, MKK 4和MKK 6激活。这些MKKs又依次被不同的MKK激酶(MAPKKK)激活。不同的刺激又可导致MAPKKK的活化。MAPKs信号转导通路正是通过这种保守的三级酶促级联反应激活其下游底物,MAPKs是该信号转导通路的枢纽,激活的MAPKs通过磷酸化核转录因子、细胞骨架蛋白及酶类等,参与细胞增殖、分化、转化及凋亡的调节,并与炎症、肿瘤及其它多种疾病发生机制密切相关。 目的 1:探讨NOD小鼠中MAPKs活化与IL-12表达的关系。 2:探讨cAMP对M-CSF诱导MAPKs活化及巨噬细胞发育的影响。 方法 1:利用Ⅰ型糖尿病(T1DM)动物模型NOD小鼠,研究了MAPKs在IL-12主要产生细胞中的磷酸化水平,分析该转导途径与T1DM中IL-12异常表达的关系,进一步探讨了MAPKs在T1DM中异常活化的机制。用C57小鼠作为对照,分别得到两种小鼠的骨髓源巨噬细胞,用姬姆萨染色法在显微镜下观察其形态的异同;用酶联免疫吸附(ELISA)分别测定IL-12的分泌水平以及MAPKs抑制因子预处理后IL-12的分泌变化;最后利用Western blot检测在不同刺激因素下,MAPKs的活化方式,比较NOD、C57小鼠的异同,为T1DM的治疗提供新的思路。 2:显微镜下观察C57小鼠骨髓源巨噬细胞的形态;Western blot分析M-CSF对MAPKs活化的影响及cAMP对M-CSF诱导MAPKs活化的影响; FACS分析cAMP对巨噬细胞发育过程中F4/80和CD14表达的影响。 结果 1: NOD小鼠巨噬细胞形态异常;ELISA结果显示NOD小鼠中IL-12异常高表达,加p38MAPK抑制剂后,表达下降;Western blot结果显示NOD小鼠巨噬细胞中,p38 MAPK信号通路异常活化,可能和MAPKKK水平有关。 2:巨噬细胞形态显示成功建立巨噬细胞发育模型;Western blot结果显示在不成熟和成熟巨噬细胞中,M-CSF均可以活化ERK、JNK和p38。随着时间的延长,cAMP抑制M-CSF诱导活化ERK、JNK和p38。FACS结果显示用cAMP预处理的巨噬细胞,其发育成熟能力减弱。 结论 NOD小鼠中,p38MAPK异常活化诱导产生大量IL-12,可能是由于MAPKKK水平出现异常造成的;cAMP通过抑制M-CSF诱导的ERK、JNK和p38活化来调控巨噬细胞的发育。
[Abstract]:Background Mitogen-activated protein kinase (MAPKs) system is one of the oldest and most conserved signal transduction systems in evolution and plays an important role in immune response. There are three subfamilies in mammalian cells, namely extracellular signal-regulated kinase ERK1 / 2, c-Jun N-terminal kinase, JNKN / stress-activated protein kinase SAPKN, and p38 protein kinase .MAPKs activated by tripeptide sequence threonine -Xaaa- tyrosine double phosphorylation. The tripeptide motifs are different in each MAPKs: ERK (threonine-glutamic acid-tyrosine) JNK( threonine-proline-tyrosine); and p38 (threonine-glycine-tyrosine). The double phosphorylation of threonine and tyrosine was regulated by conserved protein kinase system. ERK MAPK was activated by MAPK kinase MKK) MEK1 and MEK2. JNK MAPK was activated by MKK4 and MKK7 and p38 MAPK was activated by MKK3, MKK4 and MKK6. These MKKs are activated by different MKK kinases MAPKKK in turn. Different stimuli lead to MAPKKK activation. MAPKs signal transduction pathway activates its downstream substrate MAPKs through this conserved three-level enzymatic cascade reaction. MAPKs activate MAPKs through phosphorylated transcription factors. Cytoskeletal proteins and enzymes are involved in the regulation of cell proliferation, differentiation, transformation and apoptosis, and are closely related to the pathogenesis of inflammation, tumor and other diseases. Objective 1: to investigate the relationship between MAPKs activation and IL-12 expression in nod mice. 2: to investigate the effect of camp on M-CSF induced MAPKs activation and macrophage development. Methods: the phosphorylation of MAPKs in IL-12 producing cells was studied in nod mice, an animal model of type 鈪，

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