本文選題：急性早幼粒細胞性白血病 + 柔紅霉素��； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:向HL-60柔紅霉素耐藥細胞轉(zhuǎn)入miRNA-27a和反義miRNA-125b模擬體,觀察這兩種模擬體對HL-60細胞柔紅霉素(DNR)耐藥性的影響。方法:1.用小劑量DNR誘導(dǎo)急性早幼粒細胞白血病細胞株HL-60一個月,使之產(chǎn)生DNR耐藥性;2.用脂質(zhì)體Endo Fectin TM-MAX將miRNA-27a和反義miRNA-125b模擬體(mimic)以及陰性對照(N.C.)轉(zhuǎn)入HL-60 DNR耐藥細胞中;3.孵育48小時后用倒置熒光顯微鏡觀察細胞狀態(tài),用RNA提取試劑盒(RNAiso Plus,Ta Ka Ra)提取RNA,用miRNA引物、和All-in-One?miRNA qRT-PCR試劑盒(Gene Copoeia TM公司)對轉(zhuǎn)染效果進行實時熒光定量RT-PCR驗證;4.用誘導(dǎo)劑量的DNR作用于轉(zhuǎn)入上述miRNA模擬體的HL-60耐藥細胞,并分別于加藥后48和72小時進行MTT試驗,觀察這些細胞對DNR的耐受性是否發(fā)生改變;5.用SPSS17.0對實驗結(jié)果進行統(tǒng)計分析。結(jié)果:1.HL-60 DNR耐藥細胞的產(chǎn)生:小劑量DNR對HL-60細胞用藥后的前3天在顯微鏡下觀察到大面積細胞碎片,細胞大量死亡,但連續(xù)作用一個月后,未觀察到大片的細胞碎片,細胞生長狀況良好,證實HL-60細胞對DNR已產(chǎn)生耐藥;2.熒光顯微鏡下轉(zhuǎn)染效果的判定:轉(zhuǎn)染miRNA模擬體的HL-60 DNR耐藥細胞孵育48小時后,于倒置熒光顯微鏡下可觀察到大片的熒光,表明miRNA模擬體復(fù)合物已大量轉(zhuǎn)入細胞內(nèi);3.miRNA實時熒光定量PCR的檢測結(jié)果:與陰性對照(N.C.)相比,HL-60 DNR耐藥細胞內(nèi)miRNA-27a和反義miRNA-125b模擬體含量顯著增高,差異有統(tǒng)計學(xué)意義(P0.05)。4.MTT結(jié)果:與陰性對照相比,DNR對轉(zhuǎn)染miRNA-27a模擬體的HL-60 DNR耐藥細胞的抑制率顯著升高(P0.05),而對轉(zhuǎn)染反義miRNA-125b模擬體的HL-60/DNR耐藥細胞的抑制率無明顯改變(P0.05)。結(jié)論:上調(diào)miRNA-27a的表達可降低HL-60 DNR耐藥株對DNR的耐藥性,而增加反義miRNA-125b的表達不能逆轉(zhuǎn)HL-60 DNR耐藥細胞對DNR的耐藥性。
[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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.7

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本文編號：2010398