本文選題：左旋紫草素 + 白血病K562細胞��； 參考：《延安大學(xué)》2015年碩士論文

【摘要】：實驗?zāi)康?通過對左旋紫草素對白血病K562細胞、慢性粒細胞白血病原代細胞以及正常單個核細胞的作用的初步研究,拓展左旋紫草素在治療慢性粒細胞白血病方面的潛在應(yīng)用價值。實驗材料與實驗方法:左旋紫草素購買于上海萬疆生物技術(shù)有限公司,分別選取1μmol/L、2μmol/L、3μmol/L作用于白血病K562細胞(由陜西省人民醫(yī)院血液內(nèi)科實驗室用傳統(tǒng)方法傳代培養(yǎng)),在0h、12h、24h、48h時,倒置顯微鏡和熒光顯微鏡(DAPI染色)觀察不同藥物濃度作用時細胞形態(tài)變化;臺盼藍拒染法和MTT比色法檢測不同藥物濃度作用時的細胞增殖抑制率;流式細胞術(shù)檢測不同藥物濃度細胞的凋亡率。另外收集5份臨床初診的慢性粒細胞白血病患者外周血及3份正常人外周血,分離單采后經(jīng)Ficoll密度梯度離心法分離提取獲得單個核細胞,選取2μmol/L、4μmol/L的左旋紫草素,分別作用于慢性粒細胞白血病原代細胞和正常單個核細胞,選取主要檢測時間點為0h、24h、48h,臺盼藍拒染法檢測不同藥物濃度的單個核細胞增殖抑制率;利用實時定量PCR法檢測不同藥物濃度對慢性粒細胞白血病原代單個核細胞的BCR/ABL融合基因P210表達的影響。實驗結(jié)果:1.倒置顯微鏡和熒光顯微鏡(DAPI染色)下觀察左旋紫草素作用白血病K562細胞24h后細胞的形態(tài)變化:不同濃度左旋紫草素(1μmol/L、2μmol/L、3μmol/L)作用于K562細胞24h后,對照組細胞形態(tài)完整,折光性良好;藥物濃度1μmol/L可見少數(shù)細胞發(fā)生腫脹,形態(tài)變得不規(guī)則;藥物濃度2μmol/L細胞形態(tài)更加不規(guī)則,出現(xiàn)少量凋亡小體;3μmol/L實驗組出現(xiàn)大量凋亡小體及細胞碎片;2.臺盼藍拒染法、MTT比色法檢測左旋紫草素對白血病K562細胞的增殖抑制:不同濃度的左旋紫草素(1μmol/L、2μmol/L、3μmol/L)在12h、24h、48h均能抑制K562細胞的增殖,且其抑制作用呈濃度及時間依賴性;3.流式細胞術(shù)檢測左旋紫草對白血病K562細胞的凋亡影響:不同濃度左旋紫草素(1μmol/L、2μmol/L、3μmol/L)作用K562細胞24h后流式細胞儀分析K562細胞早期及晚期凋亡率實驗組分別為(4.85±0.34)%(1μmol/L),(10.53±0.72)%(2μmol/L),(18.71±1.34)%(3uoml/L),而對照組為(3.95±0.46)%。比較不同濃度左旋紫草素處理細胞后測得的各組細胞凋亡率:實驗組(1μmol/L)與對照組凋亡率相比差異無明顯統(tǒng)計學(xué)意義(P0.05);而實驗組(2μmol/L、3μmol/L)與對照組及各實驗組間細胞的凋亡率相比差異均具有統(tǒng)計學(xué)意義(P0.05);4.臺盼藍拒染法測定左旋紫草素分別對慢性粒細胞白血病原代細胞及正常單個核細胞的影響作用:不同濃度左旋紫草素(2μmol/L、4μmol/L)分別作用于慢性粒細胞白血病原代細胞及正常單個核細胞不同時間(0h、12h、24h、48h)后利用臺盼藍拒染法進行活細胞計數(shù)并換算抑制率,發(fā)現(xiàn)μmol/L濃度級左旋紫草素可明顯抑制慢性粒細胞白血病原代細胞的增殖(P0.05),且其抑制作用呈濃度及時間依賴性;而同樣濃度的左旋紫草素對正常單個核細胞的增殖無明顯影響(P0.05);5.實時熒光定量PCR檢測慢性粒細胞白血病原代細胞BCR/ABL融合基因P210的表達:慢性粒細胞白血病原代細胞暴露于不同濃度左旋紫草素(2μmol/L、4μmol/L)24h后收集細胞進行實時定量PCR檢測,其結(jié)果顯示μmol/L濃度級左旋紫草素可明顯下調(diào)慢性粒細胞白血病原代細胞BCR/ABL融合基因P210的表達(P0.05)。實驗結(jié)論:左旋紫草素對白血病K562細胞的生長增殖有明顯抑制作用,其抑制強度呈濃度及時間依賴性;左旋紫草素在對正常單個核細胞的增殖則無明顯影響作用情況下可抑制慢性粒細胞白血病原代細胞的增殖并誘導(dǎo)其凋亡,且對其BCR/ABL融合基因P210的表達具有明顯下調(diào)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.

【學(xué)位授予單位】：延安大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R733.72

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相關(guān)期刊論文 前2條

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