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目的研究全反式维甲酸(ATRA)以不同方式联用阿糖胞苷(Ara-C)或柔红霉素(DNR),作用于白血病细胞株HL-60致细胞内氧化还原物质及凋亡相关通路蛋白的变化。方法体外培养HL-60细胞株,细胞悬液以密度5×10~5·mL~(-1)接种于24孔板中。将细胞分为7组,A组为正常对照组;B组将ATRA与Ara-C同时联合作用24 h;C组预先用ATRA作用24 h,然后改用Ara-C继续作用24h;D组预先用Ara-C作用24h,然后改用ATRA继续作用24h;E组将ATRA与DNR同时联合作用24h;F组预先用ATRA作用24h,然后改用DINR继续作用24h;G组预先用DNR作用24h,然后改用ATRA继续作用24h。MTT法测定细胞活力,根据试剂盒说明书测定细胞内总SOD酶活力和GSH水平,Westem印迹检测唯BH3域蛋白Bim表达量及MAPK信号通路磷酸化水平。结果细胞抑制率为序贯给药组(C、D、G、F组)高于同时给药组(B、E组),在序贯给药组中为先化疗药后ATRA组(D、G组)高于先ATRA后化疗药(C、F组)。与A组相比,各组SOD酶活力均明显降低,其中C、F组降低最多。D、G组细胞内GSH水平明显降低,而B、C、E、F组GSH水平明显升高。唯BH3域蛋白Bim相对表达水平随细胞抑制率升高而增多。B、C、E、F组JNK磷酸化水平降低,D、G组升高;B、C、E组p38磷酸化增多;各给药组ERK磷酸化情况无显著变化。结论不同的药物联用方式能通过细胞内GSH水平、凋亡相关蛋白以及MAPK信号通路来影响HL-60的凋亡水平。
Objective To investigate the effects of all-trans retinoic acid (ATRA) on the intracellular redox species and apoptosis of leukemia cell line HL-60 in combination with Ara-C or DNR Changes in relevant pathway proteins. Methods HL-60 cells were cultured in vitro. The cell suspension was inoculated into 24-well plates at a density of 5 × 10 -5 mL -1. The cells were divided into 7 groups, A group was normal control group; B group ATRA combined with Ara-C at the same time for 24 h; C group pretreated with ATRA for 24 h, then switched to Ara-C for 24 h; A group was treated with Ara-C for 24 hours and then treated with ATRA for 24 hours; Group A was treated with ATRA and DNR for 24 hours; group A was pretreated with ATRA for 24 hours and then treated with DINR for 24 hours; Group G was pretreated with DNR for 24h, Then use ATRA continue to function 24h. Cell viability was measured by MTT assay. Total intracellular SOD activity and GSH levels were determined by kit instructions. Bim expression was measured by Western blotting alone and MAPK signaling pathway phosphorylation was measured. Results The cell inhibitory rate was higher in sequential administration group (C, D, G and F groups) than in concurrent administration group (B and E groups) G group) than after the first ATRA chemotherapy drugs (C, F group). Compared with group A, the activities of SOD in each group were significantly decreased, of which C and F decreased most. GSH levels in D and G groups were significantly decreased, but GSH levels in B, C, E and F groups were significantly increased. The relative expression level of BH3-only protein Bim increased with the inhibition rate of cells. The levels of phosphorylation of JNK in groups B, C, E and F decreased, while in groups D and G, the phosphorylation of p38 increased in groups B, C and E; the phosphorylation of ERK did not change significantly in each group. Conclusion Different combinations of drugs can affect the apoptosis of HL-60 cells through intracellular GSH levels, apoptosis-related proteins and MAPK signaling pathway.