免疫抑制性受体LILRB2对白血病细胞系增殖和凋亡的影响

Effects of immune inhibitory receptor LILRB2 on proliferation and apoptosis of leukemia cell lines

目的探讨免疫抑制性受体—白细胞免疫球蛋白样受体亚家族B成员2(LILRB2)在人白血病细胞系中的表达、功能和相关机制,为白血病的靶向治疗提供新的方向。方法采用流式细胞术和Western blotting等方法确定LILRB2在THP1、U937、K562等白血病细胞系中的表达情况;以LILRB2高表达的THP1细胞为研究对象,利用特异性靶向LILRB2的shRNA慢病毒载体(带GFP标记)包装病毒并感染THP1细胞,使用流式细胞分选技术获得GFP+的稳定表达细胞株,并分别在mRNA水平和蛋白水平检测shRNA干扰效率,同时监测LILRB2敲除后不同时间点细胞增殖、凋亡等细胞命运的变化,分析LILRB2敲除后其下游调控信号改变等相关分子机制。结果 3株细胞系均表达LILRB2,其中又以THP1的表达最高;通过Real-Time PCR及Western blotting方法表明所构建的4个shRNA慢病毒干扰质粒中,shLILRB2-717及shLILRB2-1312能显著下调LILRB2的表达;抑制LILRB2在THP1细胞中的表达可导致细胞增殖明显减缓并出现大量坏死细胞。流式细胞术分析显示:shLILRB2-717和shLILRB2-1312组细胞早期凋亡率分别为(7.90±1.61)%和(24.80±1.32)%,显著高于对照组(Scramble组)的(3.96±0.64)%(P<0.05);两组细胞的晚期凋亡率分别为(13.80±0.98)%和(23.60±1.03)%,均显著高于Scramble组的(10.80±0.48)%(P<0.05);LILRB2敲除可导致SHP1和磷酸化SHP1(p-SHP1)表达的显著下调,提示LILRB2可能通过SHP1调控白血病细胞系的增殖和凋亡。结论免疫抑制性受体LILRB2可表达在多种白血病细胞系,抑制LILRB2的表达可引起THP1细胞中SHP1和p-SHP1的显著下调并导致细胞大量凋亡,揭示SHP1可能对白血病的演变具有正向调控作用。

Objective To explore the expression, function, and relevant mechanism of the immune inhibitory receptor of leukocyte immunoglobulin-like receptors subfamily B member 2 （LILRB2） in human leukemia cell lines and to provide new clues for the targeting treatment of leukemia. Methods The expressions of LILRB2 in leukemia cell lines, such as THP1, U937, and K562 were detected by the flow cytometry and Western blotting. The shRNA plasmids specific for the knockdown of LILRB2 （ with GFP tag） was constructed and by which the virus was packaged. THP1 cells with high expression of LILRB2 were then infected by the virus. Cell lines with steady expression of GFP ＋ were obtained by the flow cell sorting. The knockdown efficiency of shRNA at both mRNA level and protein level was detected. The changes of cell proliferation and apoptosis at different time points were observed and the relevant molecular mechanisms, such as changes of downstream regulatory signals, were analyzed after the knockdown of LILRB2. Results All three cell lines expressed LILRB2 and the expression level of LILRB2 of THP1 cells was the highest. The Real-Time PCR and Western blotting findings showed that among four constructed shRNAs plasmids, shLILRB2-717 and shLILRB2-1312 significantly down- regulated the expression of LILRB2. The inhibition of LILRB2 expression in THP1 cells dramatically decreased the proliferation and significantly increased the apoptosis. The results of the flow cytometry indicated that the apoptotic rates of cells of the shLILRB2-717 group and shLILRB2-1312 group at the early stage were （7.90 ±1.61） % and （24.80 ±1.32） %, much higher than （3.96 ±0.48） % of the control group （ Scramble group） （P〈0.05）. The apoptotic rates of cells of the two groups at the late stage were （13.80 ±0.98） % and （23.60±1.03） %, much higher than （10.80 ±0.48） % of the Scramble group （P〈0.05）. The knockdown of LILRB2 led to significant down-regulation of expressions of SHP1 and p-SHP1, which indicated that LILRB2 might regulate the proliferation and apoptosis of leukemia cell lines through SHP1. Conclusion Immune inhibitory receptor LILRB2 can be expressed in a variety of leukemia cell lines. The inhibition of LILRB2 expression can lead to significant down-regulation of SHP1 and p-SHP1 in THP1 ceils and mass apoptosis, which suggests that SHP1 may positively regulate the development of leukemia.