miRNA-27a和反义miRNA-125b模拟体对HL-60细胞株耐药性的影响

发布时间：2018-06-12 17:02

本文选题：急性早幼粒细胞性白血病 + 柔红霉素　；参考：《广西医科大学》2017年硕士论文

【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(P0.05)。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(P0.05),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(P0.05)。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。
[Abstract]:Aim: to investigate the effects of miRNA-27a and antisense miRNA-125b mimics on DNR resistance in HL-60 cells. Method 1: 1. Low dose DNR was used to induce acute promyelocytic leukemia cell line HL-60 for one month, and DNR resistance was induced. MiRNA-27a and antisense miRNA-125b mimici were prepared by liposome Endo Fectin TM-MAX and negative control group (N. C.) It was transformed into HL-60 DNR resistant cells. After incubation for 48 hours, the state of the cells was observed by inverted fluorescence microscope, RNA extraction kit was used to extract RNA isoPlus Ta Ka), miRNA primer and All-in-OnemiRNA qRT-PCR kit were used to verify the transfection effect by real-time fluorescence quantitative RT-PCR. HL-60 cells were treated with induction dose of DNR, and MTT assay was performed at 48 and 72 hours after administration. The tolerance of these cells to DNR was observed. The experimental results were statistically analyzed with SPSS 17.0. Results 1. The production of HL-60 DNR resistant cells: large areas of cell fragments were observed under microscope 3 days after treatment with low dose DNR, but after one month of continuous treatment, no large pieces of cell fragments were observed. The results showed that HL-60 cells were resistant to DNR. Evaluation of transfection effect under fluorescence microscope: HL-60 DNR resistant cells transfected with miRNA mimics were incubated for 48 hours and a large amount of fluorescence was observed under inverted fluorescence microscope. These results indicate that miRNA mimic complexes have been transferred into cells in large quantities. 3. Results of real-time fluorescent quantitative PCR of miRNA: a negative control: N. C. The contents of miRNA-27a and antisense miRNA-125b mimics in HL-60 DNR resistant cells were significantly higher than those in HL-60 DNR resistant cells. Compared with the negative control, the inhibition rate of DNR on HL-60 DNR resistant cells transfected with miRNA-27a was significantly higher than that on HL-60 DNR resistant cells transfected with antisense miRNA-125b, but the inhibition rate on HL-60 / DNR resistant cells transfected with antisense miRNA-125b was not significantly changed. Conclusion: upregulating the expression of miRNA-27a can decrease the resistance to DNR of HL-60 DNR resistant cell line, but increasing the expression of antisense miRNA-125b can not reverse the resistance of HL-60 DNR resistant cell to DNR.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

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