左旋紫草素对慢性粒细胞白血病的作用研究

发布时间：2018-04-24 06:21

本文选题：左旋紫草素 + 白血病K562细胞　；参考：《延安大学》2015年硕士论文

【摘要】：实验目的:通过对左旋紫草素对白血病K562细胞、慢性粒细胞白血病原代细胞以及正常单个核细胞的作用的初步研究,拓展左旋紫草素在治疗慢性粒细胞白血病方面的潜在应用价值。实验材料与实验方法:左旋紫草素购买于上海万疆生物技术有限公司,分别选取1μmol/L、2μmol/L、3μmol/L作用于白血病K562细胞(由陕西省人民医院血液内科实验室用传统方法传代培养),在0h、12h、24h、48h时,倒置显微镜和荧光显微镜(DAPI染色)观察不同药物浓度作用时细胞形态变化;台盼蓝拒染法和MTT比色法检测不同药物浓度作用时的细胞增殖抑制率;流式细胞术检测不同药物浓度细胞的凋亡率。另外收集5份临床初诊的慢性粒细胞白血病患者外周血及3份正常人外周血,分离单采后经Ficoll密度梯度离心法分离提取获得单个核细胞,选取2μmol/L、4μmol/L的左旋紫草素,分别作用于慢性粒细胞白血病原代细胞和正常单个核细胞,选取主要检测时间点为0h、24h、48h,台盼蓝拒染法检测不同药物浓度的单个核细胞增殖抑制率;利用实时定量PCR法检测不同药物浓度对慢性粒细胞白血病原代单个核细胞的BCR/ABL融合基因P210表达的影响。实验结果:1.倒置显微镜和荧光显微镜(DAPI染色)下观察左旋紫草素作用白血病K562细胞24h后细胞的形态变化:不同浓度左旋紫草素(1μmol/L、2μmol/L、3μmol/L)作用于K562细胞24h后,对照组细胞形态完整,折光性良好;药物浓度1μmol/L可见少数细胞发生肿胀,形态变得不规则;药物浓度2μmol/L细胞形态更加不规则,出现少量凋亡小体;3μmol/L实验组出现大量凋亡小体及细胞碎片;2.台盼蓝拒染法、MTT比色法检测左旋紫草素对白血病K562细胞的增殖抑制:不同浓度的左旋紫草素(1μmol/L、2μmol/L、3μmol/L)在12h、24h、48h均能抑制K562细胞的增殖,且其抑制作用呈浓度及时间依赖性;3.流式细胞术检测左旋紫草对白血病K562细胞的凋亡影响:不同浓度左旋紫草素(1μmol/L、2μmol/L、3μmol/L)作用K562细胞24h后流式细胞仪分析K562细胞早期及晚期凋亡率实验组分别为(4.85±0.34)%(1μmol/L),(10.53±0.72)%(2μmol/L),(18.71±1.34)%(3uoml/L),而对照组为(3.95±0.46)%。比较不同浓度左旋紫草素处理细胞后测得的各组细胞凋亡率:实验组(1μmol/L)与对照组凋亡率相比差异无明显统计学意义(P0.05);而实验组(2μmol/L、3μmol/L)与对照组及各实验组间细胞的凋亡率相比差异均具有统计学意义(P0.05);4.台盼蓝拒染法测定左旋紫草素分别对慢性粒细胞白血病原代细胞及正常单个核细胞的影响作用:不同浓度左旋紫草素(2μmol/L、4μmol/L)分别作用于慢性粒细胞白血病原代细胞及正常单个核细胞不同时间(0h、12h、24h、48h)后利用台盼蓝拒染法进行活细胞计数并换算抑制率,发现μmol/L浓度级左旋紫草素可明显抑制慢性粒细胞白血病原代细胞的增殖(P0.05),且其抑制作用呈浓度及时间依赖性;而同样浓度的左旋紫草素对正常单个核细胞的增殖无明显影响(P0.05);5.实时荧光定量PCR检测慢性粒细胞白血病原代细胞BCR/ABL融合基因P210的表达:慢性粒细胞白血病原代细胞暴露于不同浓度左旋紫草素(2μmol/L、4μmol/L)24h后收集细胞进行实时定量PCR检测,其结果显示μmol/L浓度级左旋紫草素可明显下调慢性粒细胞白血病原代细胞BCR/ABL融合基因P210的表达(P0.05)。实验结论:左旋紫草素对白血病K562细胞的生长增殖有明显抑制作用,其抑制强度呈浓度及时间依赖性;左旋紫草素在对正常单个核细胞的增殖则无明显影响作用情况下可抑制慢性粒细胞白血病原代细胞的增殖并诱导其凋亡,且对其BCR/ABL融合基因P210的表达具有明显下调作用。
[Abstract]:Objective: To explore the potential value of levroshiin in the treatment of chronic myelocytic leukemia by studying the effect of levroshiin on leukemic K562 cells, primary cells of chronic myelocytic leukemia and normal mononuclear cells. Material Technology Co., Ltd., selected 1 mol/L, 2 mol/L, 3 mu mol/L, acted on leukemia K562 cells (by traditional methods used by traditional methods in the laboratory of Hematology in Shaanxi People's Hospital). At 0h, 12h, 24h, 48h, inverted microscope and fluorescence microscope (DAPI staining) were used to observe the cell morphology changes of different drug concentrations; trypan blue staining method MTT colorimetric assay was used to detect the cell proliferation inhibition rate of different drug concentrations; flow cytometry was used to detect the apoptosis rate of different drug concentration cells. In addition, the peripheral blood and 3 normal human peripheral blood were collected from 5 newly diagnosed chronic myelocytic leukemia patients, and the single nucleus was isolated and extracted by Ficoll density gradient centrifugation after separate extraction. The cells, selected 2 mu mol/L and 4 mol/L L mol/L, acted on the primary cells of chronic myelocytic leukemia and normal mononuclear cells respectively. The main detection time was 0h, 24h, 48h, and trypan blue staining method was used to detect the proliferation inhibition rate of mononuclear cells with different drug concentration, and the real-time quantitative PCR method was used to detect the chronic concentration of different drugs. The effect of BCR/ABL fusion gene P210 expression in the primary mononuclear cells of granulocytic leukemia. Experimental results: 1. inverted microscope and fluorescence microscope (DAPI staining) were used to observe the morphological changes of 24h cells after 24h in leukemic leukemic K562 cells: different concentrations of levroshiin (1 mol/L, 2, mol/L, 3 mu mol/L) acted on K562 cell 24h, The cell morphology of the control group was complete and the refraction was good, and the concentration of 1 u mol/L showed that a few cells swelled and the morphology became irregular; the drug concentration was 2 mu mol/L, the cell morphology was more irregular, a small number of apoptotic bodies appeared, and a large number of apoptotic bodies and cell fragments appeared in the 3 micron mol/L experimental group; 2. trypan blue staining method and MTT colorimetric assay were used to detect levopurple grass The proliferation inhibition of leukemic K562 cells: different concentrations of levoshikonin (1 mu mol/L, 2 mol/L, 3 mol/L) in 12h, 24h, and 48h can inhibit the proliferation of K562 cells, and their inhibitory effects were concentration and time dependent; 3. flow cytometry was used to detect the apoptosis of leukemic K562 cells in levovirus: levopurple (1 mu) (1 micron mol/) L, 2 mu mol/L, 3 mu mol/L) after 24h flow cytometry was used to analyze the early and late apoptosis rate of K562 cells (4.85 + 0.34)% (1 mu mol/L), (10.53 + 0.72)% (2 mu) (2 mu), (18.71 + 1.34)% (3uoml/L), while the control group was (3.95 + 0.46)%. The apoptosis rate of each group after different concentration of levoshikonin treated cells was compared. There was no significant difference in the apoptosis rate between the control group (1 mol/L) and the control group (P0.05), but the experimental group (2 mu mol/L, 3 mu mol/L) had statistical significance compared with the control group and the experimental group (P0.05). 4. trypan blue stain method was used to determine the primary cells and normal cells of chronic myelocytic leukemia, respectively. The effect of mononuclear cells: different concentrations of levroshiin (2 mol/L, 4 mu mol/L) acted on the cells of chronic myelocytic leukemia and the normal mononuclear cells at different time (0h, 12h, 24h, 48h), and the living cell count was counted and the inhibition rate was converted by trypan blue staining method. It was found that the concentration grade levroshiin was obviously inhibited. Promyelocytic proliferation (P0.05) of chronic myelocytic leukemia (CML), and its inhibitory effect was concentration and time dependent; and the same concentration of levroshiin had no significant effect on the proliferation of normal mononuclear cells (P0.05); 5. real time fluorescence quantitative PCR was used to detect the expression of BCR/ABL fusion gene P210 in chronic granulocytic leukemia: chronic grain size Cell leukemia primary cells were exposed to different concentrations of levroshiin (2 mol/L, 4 mol/L) 24h for real-time quantitative PCR detection. The results showed that the expression of BCR/ABL fusion gene P210 in the primary cells of chronic myelocytic leukemia (P0.05) was obviously reduced by mol/L concentration grade levroshiin (P0.05). Experimental conclusion: levopurple herb on white blood The growth and proliferation of K562 cells have a significant inhibitory effect, and the inhibitory intensity is dependent on the concentration and time dependent. In the case of the proliferation of normal mononuclear cells, levoshikonin can inhibit the proliferation and induce apoptosis of the primary cells of chronic myelocytic leukemia, and the expression of the BCR/ABL fusion gene P210 is expressed. Obviously down the effect.

【学位授予单位】：延安大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R733.72

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