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目的研究mi R-21反义寡核苷酸对地西他滨(DCA)抗白血病效应的影响及可能机制。方法将mi R-21反义核苷酸(AMO)和无义寡核苷酸(SCR)通过脂质体转染导入U937细胞,实时荧光定量PCR(q RT-PCR)验证转染效率,再分别与DCA0.5、2.0和4.0μmol·L~(-1)作用48 h。采用q RT-PCR检测人节律基因h Per3 m RNA表达,Annexin V/PI法检测凋亡,流式细胞仪检测CD14和CD11b平均荧光强度(MFI)和细胞周期。结果 AMO转染组的mi R-21表达(0.67±0.09)均低于空白组(1.10±0.06)和SCR转染组(1.04±0.08),差异有统计学意义(P<0.01)。AMO转染组的U937细胞DCA的IC50[(2.36±0.26)μmol·L~(-1)]低于空白组[(4.32±0.61)μmol·L~(-1)]和SCR转染组[(4.02±0.45)μmol·L~(-1)](P<0.01)。同一浓度下,AMO组的早期凋亡率、CD14 MFI、CD11b MFI、细胞Sub-G1期、h Per3 m RNA均高于同一浓度药物作用的空白组和SCR组,差异均具有统计学意义(P<0.01)。结论 mi R-21 AMO能显著促进DCA体外抗白血病效应,其机制可能和h Per3的激活有关。
Objective To investigate the effect of mi R-21 antisense oligonucleotide on the anti-leukemia effect of decitabine (DCA) and its possible mechanism. Methods U937 cells were transfected with mi R-21 antisense oligonucleotide (AMO) and non-sense oligonucleotide (SCR) by lipofectamine. The transfection efficiency was verified by qRT-PCR. Respectively, with DCA 0.5, 2.0 and 4.0μmol·L -1 for 48 h. QRT-PCR was used to detect the expression of human perinatal gene hPep3mRNA. Annexin V / PI assay was used to detect apoptosis. Flow cytometry was used to detect the average fluorescence intensity (MFI) and cell cycle of CD14 and CD11b. Results The expression of mi R-21 in AMO transfected group (0.67 ± 0.09) was significantly lower than that in blank transfected group (1.10 ± 0.06) and SCR transfected group (1.04 ± 0.08) (P <0.01). The IC50 [(2.36 ± 0.26) μmol·L -1] in DCA transfected U937 cells in AMO group was lower than that in blank group [(4.32 ± 0.61) μmol·L -1] and SCR transfected group [ (4.02 ± 0.45) μmol·L -1] (P <0.01). At the same concentration, the early apoptotic rate, CD14 MFI, CD11b MFI, cell sub-G1 phase and h Per3 m RNA in AMO group were significantly higher than those in blank group and SCR group with the same concentration of drug (P <0.01). Conclusion mi R-21 AMO can significantly promote the anti-leukemia effect of DCA in vitro. The mechanism may be related to the activation of h Per3.