SHP-2酪氨酸磷酸酶激活突变导致小鼠髓系异常增殖

摘要

目的:观察激活突变SHP-2酪氨酸磷酸酶是否参与髓系异常增殖的发生.方法:以野生型(WT)和SHP-2D61G/+突变型C57BL/6小鼠为研究对象,计数外周血白细胞,比较脾大小,流式细胞术检测外周血及骨髓髓系来源细胞表面标志分子(Mac-1、Gr-1),并统计外周血Mac-1和Gr-1阳性细胞率及骨髓细胞中红系 (Ter119)、髓系 (Mac-1、Gr-1)、T(CD3)、B(B220)淋巴细胞系的阳性细胞率,观察骨髓造血干/祖细胞的集落形成(CFU)能力,Western blotting检测外周血白细胞经白细胞介素 3(IL-3)和5 μg/L,刺激后磷酸化的丝氨酸/苏氨酸蛋白激酶B(p-Akt)和磷酸化的细胞外信号调节激酶(p-ERK)表达水平.结果:SHP-2D61G/+突变16周龄组小鼠较WT组外周血白细胞数增多(P<0.05),脾明显增大,同时外周血白细胞的Mac-1和Gr-1阳性细胞率增加(P<0.05),骨髓细胞中Mac-1和Gr-1的阳性细胞率也增多,但红系、淋巴细胞系的变化不明显.同时骨髓中粒-单核细胞集落形成单位(CFU-GM)较正常对照组明显增加,白细胞经IL-3刺激后Akt和ERK蛋白磷酸化水平升高.结论:SHP-2D61G/+突变可能通过MAPK及PI3K的活化而导致小鼠髓系异常增殖.%AIM: To investigate whether an activated mutant of SHP - 2 tyrosine phosphatase is involved in abnormal proliferation of murine myeloid.METHODS: Wild - type ( WT ) and SHP - 2 D61G/+ mutant mice aged 8 weeks and 16 weeks were used.The number of peripheral blood leukocytes and the spleen sizes were measured by cell counting and weighing methods,respectively.The surface markers ( Mac - 1 and Gr - 1 for myeloid, Ter119 for erythroid, CD3 for T -lymphocyte and B220 for B - lymphocyte ) of hematopoitic cells in peripheral blood and bone marrow were detected by flow cytometry.The rate of Mac - 1 or Gr - 1 positive cells in the peripheral blood and the rate of Mac - 1, Gr - 1, Ter119, CD3 or B220 positive cells in bone marrow were analyzed.The ability of colony formation unit ( CFU ) of the bone marrow was also observed by CFU assay.Finally, the expression of p - Akt and p - ERK in the peripheral blood leukocytes induced by interleukin - 3 ( IL - 3 , 5 μg/L ) was detected by Western blotting.RESULTS: The number of leukocytes in peripheral blood of 16 - week - old mice was more ( P ＜0.01 ) and the spleens were bigger in mutant SHP - 2D61G/+ mice than those in WT mice.The rate of Mac - 1 and Gr - 1 positive cells in peripheral blood leukocytes of 16 - week - old SHP - 2D61G/+ mice were dramatically increased ( P ＜ 0.05 ).Mac - 1 and Gr - 1 positive cell rates in bone marrow of SHP -2D61G/+ mice were much higher ( P ＜0.05 ) than those in WT mice and no statistic significance was found in the erythroid or lymphocyte cells.The number of CFU - GM ( represents myeloid ) was increased in mutant mice.The expression of p -Akt and p - ERK in peripheral blood leukocytes of mutant mice was significantly enhanced after stimulated with IL - 3.CONCLUSION: These results suggest that activated mutant SHP - 2 results in the disorder of mouse myeloid proliferation via MAPK and PI3K activation.