交联CD44通过Fas途径促进人肥大细胞白血病HMC-1细胞的凋亡

Crosslinked CD44 promotes apoptosis of the human mast cell leukemia HMC-1 cell via Fas pathway

目的:探讨交联CD44分子对人肥大细胞白血病HMC-1细胞株Fas表达及其介导的细胞凋亡的影响。方法:应用流式细胞术检测HMC-1细胞表面CD44及Fas的表达,并观察透明质酸(native hyaluronan,HA)、低分子质量透明质酸(fragmented hyaluronan,F-HA)处理或抗体交联CD44分子后细胞表面Fas表达的变化,进而检测PI3K、PKC及MAPK信号通路抑制剂、蛋白质合成抑制剂和肌动蛋白聚合抑制剂对CD44分子交联后细胞表面Fas表达的影响,用Northern杂交技术检测交联CD44对Fas mRNA表达的影响,用Annexin V/PI双染法检测交联CD44、HA或F-HA对Fas介导的细胞凋亡的影响。结果:HMC-1细胞高表达CD44而低表达Fas,交联CD44分子显著上调细胞表面Fas的表达(P<0.05),而Fas mRNA转录水平没有明显变化;肌动蛋白聚合抑制剂细胞松弛素B能抑制CD44上调的Fas表达(P<0.05),所检测细胞信号通路抑制剂和蛋白质合成抑制剂均未能抑制Fas表达的上调(P>0.05);F-HA和交联CD44均能够促进Fas介导的细胞凋亡(P<0.05)。结论:CD44分子可上调HMC-1细胞的Fas表达并放大Fas介导的细胞凋亡,CD44分子可能成为肥大细胞白血病治疗的新靶点。

Objective：To investigate effect of crosslinked CD44 molecule on expression of Fas in the human mast cell leukemia HMC-1 cell line and its mediated-apoptosis of the cell. Methods： Using flow cytometry assay, expressions of the CD44 and Fas on surface of the HMC-1 cell were detected, and changes of Fas expression on the cell surface after treatment with native hyaluronan （HA） and crosslinking the CD44 molecule with anti-CD44 antibody were observed, and then effects of PKC inhibitor （H-7）, PI3K inhibitor （wortmannin）, inhibitors of MAPK signaling pathway, inhibitor of protein synthesis （CHX） and inhibitor of actin polymerization （cytochalasin B, CB） on expression of Fas on the surface of the cells with the crosslinked CD44 were tested. Effect of the crosslinked CD44 on expression of Fas mRNA was detected by Northern hybridization assay. With Annexin V/PI double staining, effect of the crosslinked CD44, HA or F-HA on Fas-mediated cell apoptosis was examined. Results： In the HMC-1 cell, expression of the CD44 was high and expression of Fas was low. The crosslinked CD44 molecule significantly enhanced expression of Fas on the cell surface （P〈0.05）, but transcription level of Fas mRNA did not obviously changed. Inhibitor of actin polymerization （CB） could inhibit up-regulation of Fas expression by the CD44 （P〈0.05）. Inhibitors of cell signaling pathway and protein synthesis which were detected couldn’t inhibit up-regulation of Fas expression （P〈0.05）. All of F-HA and the crosslinked CD44 could promote Fas-mediated cell apoptosis. Conclusion： The crossliking of CD44 up-regulates Fas expression in the HMC-1 cell, and enhances Fas-mediated cell apoptosis. The CD44 molecule could become a novel target for treatment of the mast cell leukemia.