Gab2在SHP-2酪氨酸磷酸酶激活突变所致小鼠髓系异常增殖中的作用

摘要

目的:观察SHP-2信号通路中关键接头蛋白Gab2对SHP-2激活突变引发的小鼠髓系异常增殖是否具有调控作用.方法:用Gab2-/-和SHP-2D61G/+模型小鼠建立4种基因型(SHP-2+/+、Gab2-/-、SHP-2 D61g/+和SHP-2D61G/+/Gab2-/-)小鼠,解剖分析其脾大小,外周血白细胞计数,流式细胞术检测外周血及骨髓髓系细胞表面标志分子Mac-1和Gr-1并计数Mac-1和Gr-1阳性髓系细胞比例,骨髓造血干/祖细胞集落形成实验检测小鼠造血干细胞或祖细胞对细胞因子反应性,Western blotting和免疫沉淀实验检测骨髓来源肥大细胞经IL-3刺激后磷酸化蛋白激酶B (p-Akt)和磷酸化胞外信号调节激酶(p-ERK)的活化水平,以及Gab2与SHP-2蛋白的结合情况.结果:敲除Gab2后显著减轻SHP-2激活突变导致的小鼠髓系增殖表型,主要表现在:脾指数减小,外周血白细胞减少,小鼠髓系来源的Mac-1和Gr-1阳性细胞比例降低.与SHP-2D61G/+小鼠相比,经IL-3刺激后,骨髓细胞的集落形成能力显著降低；骨髓来源肥大细胞内p-ERK和p-Akt表达明显下调,SHP-2D61G/+/ab2-/-小鼠肥大细胞内无Gab2与SHP-2结合.结论:敲除Gab2可以明显减轻SHP-2D61G/+激活突变导致的小鼠髓系异常增殖,这种减轻作用可能与SHP-2无法与Gab2结合而导致下游信号途径ERK和Akt活化减弱有关.%AIM:To investigate whether Gab2,the key adapter protein in the SHP-2 signaling pathway,is involved in mouse myeloid abnormal proliferation induced by SHP-2D61G/+ mutation.METHODS:Four kinds of mouse model genotyped as SHP-2 +/+,Gab2-/-,SHP-2D61G/+ and SHP-2D61G/+ / Gab2-/-were generated from crossbreeding of Gab2-/-mice and SHP-2D61G/+ mice.The mouse spleen size was analyzed.The number of peripheral blood leukocytes was counted by cell counting and the percentage of Mac-1 or Gr-1 positive myeloid cells in the bone marrow was detected by flow cytometry.The proliferation ability of bone marrow hematopoietic stern/progenitor cells in response to cytokines was assayed by colony formation.The expression of p-ERK and p-Akt and the binding capacity of SHp-2 with Gab2 in the bone marrow-derived mast cells stimulated with IL-3 were detected by Western blotting and immunoprecipitation.RESULTS:The phenotype of myeloproliferative disorder,such as enlarged spleen size,increased leukocyte number and high percentage of myeloid cells,in SHP-2D61G/+ mutant mice was found,and was dramatically improved in SHP-2D61G/+/Gab2-/-double mutation mice.Furthermore,compared with SHP-2D61c/+ mutation mice,significandy decreased colony formation ability of the bone marrow cells with IL-3 stimulation was observed in SHP-2D61G/+/G,ab2-/-double mutation mice.A reduced phosphorylation level of ERK/Akt,and SHP-2 without binding of Gab2 were found in SHP-2D61G/+/Gab2-/-bone marrow-derived mastells with IL-3 stimulation.CONCLUSION:Gab2 knockout significantly reduces mouse myeloid abnormal proliferation induced by SHP-2D61G/+ mutation.The molecular mechanism may be associated with reduced binding of SHP-2D61G/+ under Gab2 knockout,and further weakened the activation of downstream signaling pathways of ERK and Akt.