Objective: RS4;11 cell line was used to establish BCL-2 inhibitor-resistant cell lines of B-cell acute lymphoblastic leukemia (B-ALL) and explore the possible mechanisms of drug resistance.
Methods: RS4;11 cell line was continuously induced and cultured by low and ascending concentrations of BCL-2 inhibitors navitoclax and venetoclax to construct navitoclax-resistant cell line RS4;11/Nav and venetoclax-resistant cell line RS4;11/Ven. The cell viability was detected by MTT assay, and the cell apoptosis was detected by flow cytometry. Differentially expressed genes (DEGs) between RS4;11 drug-resistant cell lines and parental cell line were detected by transcriptome sequencing technology (RNA-seq), and mRNA expression levels of DEGs between drug-resistant cell lines and parental cell line were detected by real-time PCR (RT-PCR). Western blot was used to detect the expression levels of BCL-2 family anti-apoptotic proteins in drug-resistant cell lines and parental cell line.
Results: The drug-resistant cell lines RS4;11/Nav and RS4;11/Ven were successfully established. The resistance index (RI) of RS4;11/Nav to navitoclax and RS4;11/Ven to venetoclax was 328.655±47.377 and 2 894.027±300.311, respectively. The results of cell apoptosis detection showed that compared with the drug-resistant cell lines, RS4;11 parental cell line were significantly inhibited by BCL-2 inhibitors, while the apoptosis rate of drug-resistant cell lines was not affected by the drugs. Western blot assay showed that the expression of anti-apoptotic proteins of BCL-2 family did not increase significantly in drug-resistant cell lines. RNA-seq, RT-PCR and Western blot assays showed that the expression of EP300 in drug-resistant cell lines was significantly higher than that in parental cell line (P <0.05).
Conclusion: Drug-resistant B-ALL cell lines could be successfully established by exposing RS4;11 cell line to the ascending concentration of BCL-2 inhibitors, and the drug resistance mechanism may be related to the overexpression of EP300.
题目: 急性B淋巴细胞白血病BCL-2抑制剂耐药细胞系的构建及耐药机制研究.
目的: 利用急性B淋巴细胞白血病（B-ALL）细胞系RS4;11构建对BCL-2抑制剂耐药的耐药细胞系，并探讨其可能的耐药机制。.
方法: 采用BCL-2抑制剂navitoclax和venetoclax小剂量低浓度递增的方法间歇诱导RS4;11细胞系，构建RS4;11/Nav和RS4;11/Ven耐药细胞系，通过MTT法检测不同药物浓度下细胞的存活率，流式细胞术检测细胞凋亡，转录组测序技术（RNA-seq）检测RS4;11耐药细胞系和亲本细胞系中的差异表达基因（DEGs），RT-PCR检测耐药细胞系与亲本细胞系中差异表达基因的mRNA表达水平，Western blot检测耐药细胞系和亲本细胞系中BCL-2家族抗凋亡蛋白的表达水平。.
结果: 成功构建了对BCL-2抑制剂耐药的耐药细胞系RS4;11/Nav和RS4;11/Ven，RS4;11/Nav对navitoclax的耐药指数为328.655±47.377，RS4;11/Ven对venetoclax的耐药指数为2 894.027±300.311。流式细胞术检测细胞凋亡发现，相比耐药细胞系，RS4;11亲本细胞系明显被BCL-2抑制剂抑制，而耐药细胞系的凋亡率基本未受药物的影响。Western blot检测结果表明，BCL-2家族抗凋亡蛋白在耐药细胞系中的表达无明显增多。RNA-seq、RT-PCR和Western blot检测发现EP300在耐药细胞系中的表达较亲本细胞系明显增高（P <0.05）。.
结论: 小剂量低浓度递增间歇诱导法可成功构建对BCL-2抑制剂耐药的B-ALL细胞系，并且其耐药机制可能与EP300的表达上调有关。.
Keywords: B-cell acute lymphoblastic leukemia; BCL-2 inhibitors; drug-resistant cell line; RNA-seq; EP300.