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目的:观察骨肉瘤细胞MG-63在体外诱导逆转过程中DNA甲基转移酶(DNA methyltransferase,Dnmt)的变化,探讨Dnmt与骨肉瘤细胞逆转的相关性及其作用。方法:体外用全反式维甲酸(all trans-retinoic acid,ATRA)对MG-63细胞诱导分化,透射电镜观察瘤细胞的超微结构;MTT检测细胞的增殖能力;RT-PCR法检测细胞Dnmt和增殖核抗原(proliferationcell nuclear antigen,PCNA)mRNA的表达;以流式细胞仪检测药物作用前后细胞周期的改变。结果:经诱导剂处理后,MG-63细胞的生长明显受到抑制,D值从0.8405±0.1305下降到0.6147±0.0427(P<0.05),细胞形态呈良性分化;与对照组相比,Dnmt和PCNA mRNA的表达随着作用时间的延长均降低,细胞周期阻滞于G1期。结论:ATRA对人骨肉瘤细胞株有诱导分化作用,PCNA mRNA表达水平和细胞周期的进程与Dnmt的调节密切相关,抑制Dnmt基因的表达是ATRA诱导骨肉瘤细胞分化的重要机制之一。
OBJECTIVE: To observe the changes of DNA methyltransferase (Dnmt) during the induction of osteosarcoma cell line MG-63 in vitro and to explore the correlation between Dnmt and the reversal of osteosarcoma cells. METHODS: MG-63 cells were induced to differentiate by all-trans retinoic acid (ATRA) in vitro. The ultrastructure of the tumor cells was observed by transmission electron microscopy. The proliferation of MG-63 cells was detected by MTT assay. The expression of Dnmt And proliferating cell nuclear antigen (PCNA) mRNA expression were detected by flow cytometry before and after the drug effect on cell cycle changes. Results: The growth of MG-63 cells was significantly inhibited by the inducer, the D value decreased from 0.8405 ± 0.1305 to 0.6147 ± 0.0427 (P <0.05), and the cell morphology was benign. Compared with the control group, Dnmt and PCNA The mRNA expression decreased with the prolongation of action time, and the cell cycle arrest in G1 phase. CONCLUSION: ATRA induces differentiation of human osteosarcoma cell lines. The expression of PCNA mRNA and cell cycle progression are closely related to the regulation of Dnmt. Suppression of Dnmt gene expression is one of the important mechanisms of ATRA-induced osteosarcoma cell differentiation.