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    泛素化连接酶c-Cbl对急性髓系白血病细胞增殖分化能力的影响

    Effect of ubiquitinated ligase c-Cbl on proliferation and differentiation of acute myeloid leukemia cells

      摘要
    • 摘要:
      目的 探讨泛素化连接酶c-Cbl对急性髓系白血病细胞株THP-1细胞(人急性单核细胞白血病细胞)增殖分化能力的影响。
      方法 构建并筛选出稳定转染HBLV-h-CBL-ZsGreen-PURO敲低和HBLV-h-CBL-HA-BSD过表达病毒载体的THP-1细胞，分为4组: NC组(c-Cbl敲低组对照)、S3组(c-Cbl敲低组)、EV组(c-Cbl过表达组对照)、c-Cbl组(c-Cbl过表达组)。采用Cell Counting Kit-8(CCK-8试剂盒)和细胞周期与细胞凋亡检测试剂盒检测稳转细胞的增殖和凋亡。采用蛋白免疫印迹技术检测基因敲低或过表达细胞的凋亡相关蛋白(Bcl-2、caspase-9、cleaved-caspase-9)的表达情况。采用硝基四氮唑蓝(NBT)还原实验、瑞氏吉姆萨染色、流式细胞术检测表面分化抗原CD11b表达差异。采用蛋白免疫印迹技术检测PU.1蛋白表达情况来研究稳转细胞的分化能力。
      结果 转染慢病毒后，与NC组相比，S3组中c-Cbl基因的蛋白表达水平下降；与EV组相比，c-Cbl组中c-Cbl基因的蛋白表达水平上升。CCK8和流式细胞术结果表明c-Cbl能够促进THP-1的增殖(P < 0.01)。蛋白免疫印迹结果表明c-Cbl能够抑制凋亡蛋白的表达，并且促进Bcl-2蛋白表达。NBT还原实验结果表明c-Cbl敲除后NBT阳性细胞增多，c-Cbl过表达后阳性细胞减少(P < 0.01)。瑞氏吉姆萨染色结果显示，c-Cbl敲低后细胞核分化成肾型、蚕豆型。流式细胞术结果表明c-Cbl敲除后CD11b表达增多，过表达后CD11b表达减少。蛋白免疫印迹结果也显示c-Cbl敲除后PU.1蛋白表达增多，过表达后减少。
      结论 c-Cbl能够促进THP-1细胞的增殖，抑制其凋亡和分化。

       

      Abstract:
      Objective To investigate the effect of ubiquitylated ligase c-Cbl on the proliferation and differentiation of acute myeloid leukemia cell line THP-1.
      Methods THP-1 cells stably transfected with HBLV-h-CBL-ZsGreen-PURO knockdown and HBLV-h-CBL-HA-BSD overexpression viral vectors were constructed and screened.Cells were divided into four groups including NC group (c-Cbl knockdown group control), S3 group (c-Cbl knockdown group), EV group (c-Cbl overexpression group control) and c-Cbl group (c-Cbl overexpression group).Cell Counting Kit-8 (CCK-8 kit) and cell cycle and apoptosis detection kit were used to detect the proliferation and apoptosis of the stable cells.The expression of apoptosis-related proteins (Bcl-2, Caspase-9, Cleaved-Caspase-9) in the gene knockdown or overexpression cells was detected by Western blotting.Nitrotetrazolium blue chloride (NBT) reduction test, Wright-Giemsa staining and flow cytometry were used to detect the expression of surface differentiation antigen CD11b.The expression of PU.1 protein was detected by Western blotting to study the differentiation ability of stably transformed cells.
      Results After transfection with lentivirus, the levels of c-Cbl gene in S3 group were decreased compared with NC group.Compared with EV group, levels of c-Cbl gene were increased in c-Cbl group.CCK8 and flow cytometry showed that c-Cbl could promote THP-1 proliferation(P < 0.01).Western blotting results showed that c-Cbl could inhibit the expression of apoptotic protein and promote the expression of Bcl-2 protein.The results of NBT reduction test showed that the number of NBT-positive cells increased after c-Cbl knockout, and the number of positive cells decreased after c-Cbl overexpression(P < 0.01).The results of Wright-Giemsa staining showed that c-Cbl knockdown made the nucleus differentiate into kidney type and broad bean type.Flow cytometry showed that CD11b expression increased after c-Cbl knockout, and decreased after overexpression.Western blotting results also showed that the expression of PU.1 protein increased after c-Cbl knockout, but decreased after overexpression.
      Conclusions C-Cbl can promote the proliferation of THP-1 cells and inhibit its apoptosis and differentiation.

       

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