【作者】 陈晨；

【导师】 李卫；

【作者基本信息】 广西医科大学 ， 儿科学， 2017， 硕士

【摘要】 目的:向HL-60柔红霉素耐药细胞转入miRNA-27a和反义miRNA-125b模拟体,观察这两种模拟体对HL-60细胞柔红霉素(DNR)耐药性的影响。方法:1.用小剂量DNR诱导急性早幼粒细胞白血病细胞株HL-60一个月,使之产生DNR耐药性;2.用脂质体Endo Fectin TM-MAX将miRNA-27a和反义miRNA-125b模拟体(mimic)以及阴性对照(N.C.)转入HL-60 DNR耐药细胞中;3.孵育48小时后用倒置荧光显微镜观察细胞状态,用RNA提取试剂盒(RNAiso Plus,Ta Ka Ra)提取RNA,用miRNA引物、和All-in-One?miRNA qRT-PCR试剂盒(Gene Copoeia TM公司)对转染效果进行实时荧光定量RT-PCR验证;4.用诱导剂量的DNR作用于转入上述miRNA模拟体的HL-60耐药细胞,并分别于加药后48和72小时进行MTT试验,观察这些细胞对DNR的耐受性是否发生改变;5.用SPSS17.0对实验结果进行统计分析。结果:1.HL-60 DNR耐药细胞的产生:小剂量DNR对HL-60细胞用药后的前3天在显微镜下观察到大面积细胞碎片,细胞大量死亡,但连续作用一个月后,未观察到大片的细胞碎片,细胞生长状况良好,证实HL-60细胞对DNR已产生耐药;2.荧光显微镜下转染效果的判定:转染miRNA模拟体的HL-60 DNR耐药细胞孵育48小时后,于倒置荧光显微镜下可观察到大片的荧光,表明miRNA模拟体复合物已大量转入细胞内;3.miRNA实时荧光定量PCR的检测结果:与阴性对照(N.C.)相比,HL-60 DNR耐药细胞内miRNA-27a和反义miRNA-125b模拟体含量显著增高,差异有统计学意义(P<0.05)。4.MTT结果:与阴性对照相比,DNR对转染miRNA-27a模拟体的HL-60 DNR耐药细胞的抑制率显著升高(P<0.05),而对转染反义miRNA-125b模拟体的HL-60/DNR耐药细胞的抑制率无明显改变(P>0.05)。结论:上调miRNA-27a的表达可降低HL-60 DNR耐药株对DNR的耐药性,而增加反义miRNA-125b的表达不能逆转HL-60 DNR耐药细胞对DNR的耐药性。更多还原

【Abstract】 OBJECTIVE: The miRNA-27 a and antisense miRNA-125 b mimics were transfected into HL-60 daunorubicin-resistant cells to observe the effects of these two mimetics on the resistance of daunorubicin(DNR)to HL-60 cells.METHODS:1.A small dose of DNR induced acute promyelocytic leukemia cell line HL-60 for one month to produce DNR drug resistance.2.The antisense strand mimic of miRNA-125 b and the sense strand mimic miRNA-27 a and negative control(N.C.)were transfected into HL-60/DNR-resistant cells by liposome Endo Fectin TM-MAX.3.Observe the transfected cell status by fluorescence microscopy after incubation for 48 hours.extract the RNA by Ta Ka Ra RNA extraction kit(RNAiso Plus),verify the transfection according to All-in-One ? miRNA q RT-PCR kit which was provided by Gene Copoeia TM,and the miRNA primer sequences were provided by Shanghai Bioengineering Co.,Ltd..4.With low doses of daunorubicin acting on the above cells,MTT assay was performed to the cells after 48 and 72 hours.Observe whether these cells are resistant to DNR.Results: 1.The state of HL-60 sensitive cells: there were a large area of cell debris in the first 3 days under the microscope,then we did not find significant cell debris after a month,confirmed that HL-60 cells significantly improved tolerance to DNR,cells had been resistant to DNR;2.Evalue the efect by fluorescence microscope: Transfected HL-60/DNR-resistant cells with miRNA mimics and then incubated for 48 hours,there were exsist fluorescence in large number of cells,indicated that the mix of miRNA mimic complex had been expressed intracellularly;3.The results of real-time quantitative PCR showed that the miRNA-27 a and antisense miRNA-125 b were significantly increased,compared with the cells which was transfected with negative control(P <0.05).4.The results of MTT assay showed that the inhibitory rate of daunorubicin on HL-60 / DNR-resistant cells transfected with miRNA-27 a mimics was significantly higher than that of the miRNA-NC transfected group(P<0.05),the inhibitory rate of HL-60 / DNR cells transfected with antisense miRNA-125 b was not significantly changed(P> 0.05).Conclusion: Upregulation of miRNA-27 a can decrease the resistance of HL-60 / DNR-resistant strains to daunorubicin,and it is proved that the overexpression of antisense miRNA-125 b can not reverse the resistance of HL-60 / DNR-resistant strains to daunorubicin.更多还原