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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 apoptosis induced by proteasome inhibitor bortezomib (Bor) in acute erythroleukemia cell line (TF1) and acute promyelocytic leukemia cell line (NB4) and its effect on SALL4 gene expression. Methods Cell proliferation was detected by MTT assay. Cell apoptosis was detected by flow cytometry. SALL4 protein expression was detected by immunocytochemistry. SALL4 RNA expression was detected by real-time fluorescence quantitative PCR. SALL4 protein expression was detected by Western Blotting. Results MTT results showed that bortezomib could inhibit the proliferation of both cells in a time and dose dependent manner. The IC50 of TF1 cells and NB4 cells for 48 h were (29.15 ± 0.55) and (30.55 ± 0.74) nmol.L-1, respectively. Flow cytometry showed that apoptosis rate increased with the increase of bortezomib concentration in a dose-dependent manner. Immunocytochemistry showed that both cells expressed SALL4 protein and localized in the nucleus. Real-time quantitative PCR results showed that after treated with different concentrations of Bor (10, 30 and 50 nmol.L-1) for 24 h, SALL4 RNA (P <0.05). The expression of SALL4 decreased to 45.11% (TF1) and 69.77% (NB4) in the control group after treatment with 50 nmol.L-1 Bor. The difference was statistically significant (P <0.05). Western Blotting results showed that Bor inhibited the expression of SALL4B protein in both cells in a dose-and time-dependent manner. Conclusion Bortezomib can inhibit the proliferation and promote the apoptosis of TF1 and NB4 cells and inhibit the expression of SALL4 gene.