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近年来,鸟氨酸脱羧酶抗酶(OAZ)作为肿瘤治疗的潜在靶点备受关注.本文研究了OAZ1基因过表达对慢粒白血病K562细胞红系分化的作用.构建框移位点突变的OAZ1过表达慢病毒载体pLVX-Neo-OAZ1-IRES-ZsGreen,包装病毒并感染K562细胞,Western印迹验证其过表达效果.FACS检测细胞分化标志物CD71和GPA,结合联苯胺染色分析细胞红系分化情况.对比氯化高铁血红素(hemin)诱导组,实时RT-PCR检测与K562细胞红系分化、癌变的关键基因(GATA1、BCR/ABL、TGFβ)转录水平,对OAZ1诱导分化的机制进行初步探索.结果表明,慢病毒过表达载体及K562细胞过表达体系构建成功.OAZ1过表达后细胞红系分化标志物CD71+/GPA+为(11.22±2.09)%,与对照组(4.07±1.04)%、空病毒组(1.79±2.36)%相比差异极显著(P<0.01);联苯胺蓝染阳性率为(14.037±0.083)%,与对照组、空病毒组比较,差异也极显著(P<0.01).定量分析结果提示,相对于GATA1、BCR/ABL基因mRNA转录水平的影响,OAZ1对TGFβ基因的作用更为明显.为此推断,OAZ1基因可诱导白血病K562细胞向成熟红系方向分化,其作用机制可能与TGFβ信号转导通路相关.
In recent years, ornithine decarboxylase enzyme (OAZ) as a potential target for cancer treatment of concern .In this paper, OAZ1 gene overexpression on erythroid differentiation of chronic myeloid leukemia K562 cells role in the construction of the frameshift mutation OAZ1 overexpression lentiviral vector pLVX-Neo-OAZ1-IRES-ZsGreen, packaging virus and infected K562 cells, Western blot to verify its overexpression.FACS detection of cell differentiation markers CD71 and GPA, combined with benzidine staining erythroid differentiation (GATA1, BCR / ABL and TGFβ) in erythroid differentiation and carcinogenesis of K562 cells were detected by real time RT-PCR, and the mechanism of OAZ1 induced differentiation was compared with that of hemin induction group The results showed that the lentiviral overexpression vector and the over-expression system of K562 cells were successfully constructed.The erythroid differentiation marker CD71 + / GPA + was (11.22 ± 2.09)%, which was significantly higher than that of the control group (4.07 ± 1.04)%, (1.79 ± 2.36)%, the positive rate of benzidine blue staining was (14.037 ± 0.083)%, which was significantly higher than that of the control group and the empty virus group (P < 0.01) .Quantitative analysis of the results suggested that, relative to GATA1, BCR / ABL groups The effect of OAZ1 on TGFβ gene is more obvious due to the transcriptional level of mRNA.Therefore, we infer that OAZ1 gene can induce leukemia K562 cells to differentiate into mature erythroid lineage, and its mechanism may be related to TGFβ signaling pathway.