硼替佐米诱导急性髓性白血病细胞凋亡及对SALL4基因表达的影响

Induction of Acute Myelogenous Leukemia Cell Apoptosis by Bortezomib and Its Effect on SALL4 Gene Expression

目的探讨蛋白酶体抑制剂硼替佐米(bortezomib、Bor)诱导急性红白血病细胞株(TF1)和急性早幼粒白血病细胞株(NB4)凋亡及其对SALL4基因表达的影响。方法 MTT法检测细胞的增殖情况;流式细胞术检测细胞凋亡;免疫细胞化学检测SALL4蛋白表达;实时荧光定量PCR检测SALL4 RNA的表达变化;Western Blotting检测SALL4蛋白的表达情况。结果 MTT结果显示,硼替佐米能够抑制两种细胞的增殖,呈时间和计量依赖性,TF1细胞和NB4细胞48 h IC50分别为(29.15±0.55)和(30.55±0.74)nmol.L-1;流式细胞术结果显示,细胞凋亡率随着硼替佐米浓度的增加而增加,呈剂量依赖性。免疫细胞化学显示两种细胞均表达SALL4蛋白,定位于细胞核;实时荧光定量PCR结果表明,经不同浓度Bor(10,30,50 nmol.L-1)处理24 h后,两种细胞的SALL4 RNA均出现了不同程度的下调,50 nmol.L-1 Bor作用后,SALL4基因表达下降为对照组的45.11%(TF1)和69.77%(NB4),差异均具有统计学意义(P<0.05);Western Blotting结果表明,Bor能够抑制两种细胞中SALL4B蛋白的表达,具有时间剂量依赖性。结论硼替佐米能够抑制TF1、NB4细胞的增殖并促进细胞凋亡,同时抑制SALL4基因表达。

OBJECTIVE To investigate the proteasome inhibitor bortezomib induces acute myelogenous leukemia cell lines TF1 and NB4 apoptosis and its effect on SALIA gene expression. METHODS Cell proliferations were analyzed by MTT assay. Flow cytometry was used to analyze cell apoptosis rate. SALL4 protein was detected by immunocytochemistry. The expressions of SALL4 gene were detected by Real-time PCR. SALLA proteins in two cell lines were detected by Western Blotting. RESULTS MTF assay showed bortezomib inhibited the proliferations of two cell lines in a time-and-does manner. TFl and NB4 ceils＇ 48 h IC50 were （29. 15±0. 55 ） and （30. 55±0. 74） nmol·L^-1respectively. Flow cytometry showed hortezomib could induce apoptosis of two cell lines in a dose-dependent manner. Immunocytochemistry analysis revealed that both of two cell lines expressed SALL4 proteins which located in cell nu- cleus. Real-time PCR demonstrated that SALL4 genes were down-regulated after cells were treated by different concentrations（ 10,30, 50 nmol·L-1） of bortezomib for 24 h, and bortezomib 50 nmol·L^-1 groups＇ genes were down-regulated to 45. 11% （TF1） and 69.77% （NB4） respectively comparing with the control groups （P 〈 0. 05）. Western blotting revealed that both of the cell lines expressed SALL4B proteins and which could be inhibited by bortezomib in a time-and-does manner. CONCLUSION Bortezomib can significantly inhibit two cell lines proliferation and induce apoptosis, meanwhile down-regulate the expressions of SALIA gene.