CD44抗体HI44a对急性髓系白血病细胞株HL-60细胞体外增殖、分化作用的研究

目的探讨CD44抗体HI44a对急性髓系白血病细胞株HL-60细胞增殖、分化的作用机制。方法以急性髓系白血病细胞株HL-60为研究对象,以ATRA为阳性对照,以同型抗体IgG2a为阴性对照,以HI44a作为实验组,应用台盼蓝拒染法检测HI44a对HL-60细胞增殖的影响,瑞氏染色法及流式细胞学检测HI44a对HL-60细胞分化的影响,RT-PCR方法检测HI44a处理HL-60细胞前后TGFβ1和TGFβR1基因表达变化,应用荧光定量RT-PCR法检测HI44a处理HL-60细胞前后髓系分化相关的细胞因子及受体信号通路(GM-CSFRα、OPN)基因表达变化,以及DNA序列分析法检测HI44a处理HL-60细胞前后OPN异构体表达改变。采用酶联免疫吸附试验(ELISA)检测实验组与对照组培养上清液的OPN水平。结果经HI44a作用后,HL-60细胞增殖受抑;细胞形态变化表现为体积变大,核固缩,核浆比例降低,核仁减少,出现核分叶等特征;细胞表面分化抗原CD15表达上调;HL-60细胞的GM-CSFRα及TGFβ1 mRNA表达水平增高,而OPN mRNA表达水平降低,TGFβR1 mR-NA表达水平无变化;OPN序列分析结果表明HI44a作用后OPN的3种异构体的组成发生改变,HI44a处理后的OPN水平明显下降。结论 CD44抗体HI44a通过上调GM-CSFRα、TGFβ1及下调OPN mRNA表达水平,减少OPN的分泌,抑制HL-60细胞增殖,诱导细胞分化。

The effect of CD44 antibody-HI44a on proliferation and differentiation of HL-60 cells

Aim To investigate the effect of CD44 antibody-HI44a on proliferation and differentiation of HL-60 cells.Methods The effect of HI44a on proliferation and differentiation in HL-60 cells was detected by trypan blue staining method,wright staining and flow cytometry,taking ATRA as positive control and IgG2a as isotype control.The expression of TGF-β1 and TGF-βR1 was detected by RT-PCR,the expression of IL-3Rα,GM-CSFRα,Hβc and OPN was detected by real-time quantitative RT-PCR.The full-length cDNA of osteopontin was sequenced by DNA sequence analysis.The OPN level of conditioned media was analyzed by ELISA.Results The proliferation of HL-60 cells was inhibited by HI44a;the cell morphological change presented the reduced nucleolus,nuclear sublobe and so on;the expression level of GM-CSFRα and TGF-β1 mRNA was upregulated,while the expression level of osteopontin mRNA was downregulated,and the expression level of TGF-βR1 mRNA expression did not show obvious change;DNA sequence analysis showed that the composition of three osteopontin isoforms changed.The OPN level in HI44a group was significantly lower than that in HL-60 group.Conclusions CD44 antibody HI44a can inhibit HL-60 cell proliferation,and induce the differentiation of HL-60 cells by upregulating the expression of GM-CSFRα,TGF-β1 mRNA,downregulating the expression of OPN mRNA and reducing the secretion of OPN.