本文選題：MAPKs + 小鼠��； 參考：《河南大學(xué)》2008年碩士論文

【摘要】： 背景 絲裂原活化蛋白激酶(MAPKs)系統(tǒng)是進化中最古老最保守的信號轉(zhuǎn)導(dǎo)系統(tǒng)之一,在免疫應(yīng)答過程中起重要作用。在哺乳動物細胞中主要有三個亞家族,分別為細胞外信號調(diào)節(jié)激酶(ERK1/2)、c-Jun N端激酶(JNK)/應(yīng)激活化蛋白激酶(SAPK)、和p38蛋白激酶。MAPKs通過三肽基序蘇氨酸-Xaa-酪氨酸發(fā)生雙磷酸化而活化。該三肽基序序列在每個MAPKs中是不同的:ERK (蘇氨酸-谷氨酸-酪氨酸);JNK(蘇氨酸-脯氨酸-酪氨酸);和p38(蘇氨酸-甘氨酸-酪氨酸)。蘇氨酸和酪氨酸的雙磷酸化被保守的蛋白激酶系統(tǒng)調(diào)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通過MKK 3, MKK 4和MKK 6激活。這些MKKs又依次被不同的MKK激酶(MAPKKK)激活。不同的刺激又可導(dǎo)致MAPKKK的活化。MAPKs信號轉(zhuǎn)導(dǎo)通路正是通過這種保守的三級酶促級聯(lián)反應(yīng)激活其下游底物,MAPKs是該信號轉(zhuǎn)導(dǎo)通路的樞紐,激活的MAPKs通過磷酸化核轉(zhuǎn)錄因子、細胞骨架蛋白及酶類等,參與細胞增殖、分化、轉(zhuǎn)化及凋亡的調(diào)節(jié),并與炎癥、腫瘤及其它多種疾病發(fā)生機制密切相關(guān)。 目的 1:探討NOD小鼠中MAPKs活化與IL-12表達的關(guān)系。 2:探討cAMP對M-CSF誘導(dǎo)MAPKs活化及巨噬細胞發(fā)育的影響。 方法 1:利用Ⅰ型糖尿病(T1DM)動物模型NOD小鼠,研究了MAPKs在IL-12主要產(chǎn)生細胞中的磷酸化水平,分析該轉(zhuǎn)導(dǎo)途徑與T1DM中IL-12異常表達的關(guān)系,進一步探討了MAPKs在T1DM中異常活化的機制。用C57小鼠作為對照,分別得到兩種小鼠的骨髓源巨噬細胞,用姬姆薩染色法在顯微鏡下觀察其形態(tài)的異同;用酶聯(lián)免疫吸附(ELISA)分別測定IL-12的分泌水平以及MAPKs抑制因子預(yù)處理后IL-12的分泌變化;最后利用Western blot檢測在不同刺激因素下,MAPKs的活化方式,比較NOD、C57小鼠的異同,為T1DM的治療提供新的思路。 2:顯微鏡下觀察C57小鼠骨髓源巨噬細胞的形態(tài);Western blot分析M-CSF對MAPKs活化的影響及cAMP對M-CSF誘導(dǎo)MAPKs活化的影響; FACS分析cAMP對巨噬細胞發(fā)育過程中F4/80和CD14表達的影響。 結(jié)果 1: NOD小鼠巨噬細胞形態(tài)異常;ELISA結(jié)果顯示NOD小鼠中IL-12異常高表達,加p38MAPK抑制劑后,表達下降;Western blot結(jié)果顯示NOD小鼠巨噬細胞中,p38 MAPK信號通路異常活化,可能和MAPKKK水平有關(guān)。 2:巨噬細胞形態(tài)顯示成功建立巨噬細胞發(fā)育模型;Western blot結(jié)果顯示在不成熟和成熟巨噬細胞中,M-CSF均可以活化ERK、JNK和p38。隨著時間的延長,cAMP抑制M-CSF誘導(dǎo)活化ERK、JNK和p38。FACS結(jié)果顯示用cAMP預(yù)處理的巨噬細胞,其發(fā)育成熟能力減弱。 結(jié)論 NOD小鼠中,p38MAPK異常活化誘導(dǎo)產(chǎn)生大量IL-12,可能是由于MAPKKK水平出現(xiàn)異常造成的;cAMP通過抑制M-CSF誘導(dǎo)的ERK、JNK和p38活化來調(diào)控巨噬細胞的發(fā)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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