本文選題：DBO + K562細胞��； 參考：《濟南大學》2017年碩士論文

【摘要】：背景慢性髓系細胞白血病(CML)是一種起源于多能干細胞的髓系惡性增殖性腫瘤,t(9;22)(q34;q11)是CML特征性染色體改變并在分子水平上導致BCR-ABL融合基因形成的。這種BCR-ABL融合基因形成的融合蛋白是一種能導致細胞轉化的多種信號蛋白相關的酪氨酸激酶。目前酪氨酸激酶抑制劑(TKI)(包括伊馬替尼,格列衛(wèi)等)已廣泛用于CML治療,使大部分CML患者生存期延長,達到細胞遺傳學或分子生物學上的緩解。隨著TKI臨床應用時間延長,TKI耐藥的問題也越來越明顯,積極探索新的藥物是一個亟待解決的問題。DBO(6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine)是一種苯并惡嗪衍生物,有研究證實其可作為哺乳動物雷帕霉素靶蛋白(m TOR)抑制劑誘導人臍靜脈內皮細胞產生細胞活性氧物質(ROS),從而誘導人臍靜脈內皮細胞發(fā)生自噬,促進其凋亡。然而,目前其抗腫瘤作用尚未廣泛研究,抗腫瘤作用的機制目前尚無定論。關于DBO是否引起白血病細胞發(fā)生自噬、凋亡的研究尚未報道。本研究以人K562細胞為研究對象,分析DBO對K562細胞的增殖及其在誘導細胞凋亡和自噬方面的影響。目的本研究旨在探討DBO對人慢性髓系白血病K562細胞的增殖、自噬與凋亡的影響。方法常規(guī)方法復蘇、傳代培養(yǎng)K562細胞,設置對照組(DMSO)、DBO處理組。處理24、48和72h后收集細胞,采用蛋白質印跡法檢測LC3蛋白的表達。細胞免疫熒光實驗檢測LC3斑點的表達;透射電鏡觀察細胞自噬現(xiàn)象。CCK-8比色法檢測K562細胞的活性和增殖能力。細胞周期實驗檢測K562細胞分裂能力。Annexin V-FITC/PI雙染流式細胞術檢測細胞凋亡。結果蛋白質印跡結果顯示,K562細胞經雷帕霉素處理后LC3-Ⅱ蛋白表達水平升高,p62蛋白表達水平降低;經50μmol/L DBO處理24、48和72 h后,LC3-Ⅱ和p62蛋白表達水平與雷帕霉素處理后結果相似,并呈時間依賴性。K562細胞經10、25和50μmol/L DBO處理后,LC3-Ⅱ蛋白表達水平升高,p62蛋白表達水平降低,呈劑量依賴性,而使用自噬抑制劑3-MA后LC3-Ⅱ蛋白表達降低,P62蛋白表達升高。細胞免疫熒光實驗顯示,DBO作用72 h后,與對照組相比,DBO處理組LC3斑點的表達量增加。透射電鏡觀察結果顯示,與對照組相比,DBO處理組細胞胞質內可見較多自噬小體和自噬溶酶體,細胞核不規(guī)則,染色質邊緣化。CCK8檢測結果顯示,DBO對K562細胞的增殖具有明顯的抑制作用,并呈現(xiàn)時間-劑量依賴性。DBO經不同濃度(10μmol/L、25μmol/L、50μmol/L、100μmol/L)處理24 h的細胞增殖抑制率分別為(0.68±0.05)%、(2.76±0.35)%、(12.64±3.90)%、(22.58±2.41)%,F=67.389,P0.001,處理組與對照組之間差異有統(tǒng)計學意義;48 h的增殖抑制率分別為(3.83±1.06)%、(6.23±1.27)%、(15.90±1.10)%、(24.50±2.51)%,F=145.738,P0.001,處理組與對照組之間差異有統(tǒng)計學意義;72h的增殖抑制率分別為(8.78±1.28)%、(21.38±1.47)%、(32.11±2.01)%、(34.27±2.59)%,F=225.820,P0.001,處理組與對照組之間差異有統(tǒng)計學意義。流式細胞術分析顯示K562細胞經DBO處理72 h后,與對照組相比,G2/M期細胞比例增加,χ2=276.706,P0.001。流式細胞術分析顯示,與對照組相比,K562細胞經DBO處理72 h后的凋亡率增加,χ2=227.384,P0.001。結論DBO可明顯抑制K562細胞生長,并與自噬和凋亡的發(fā)生有關。
[Abstract]:Background chronic myelocytic leukemia (CML) is a malignant proliferative tumor of myeloid origin derived from pluripotent stem cells. T (9; 22) (q34; Q11) is a characteristic chromosome change of CML and causes the formation of BCR-ABL fusion genes at the molecular level. The fusion protein formed by the BCR-ABL fusion gene is a variety of signal eggs that can lead to cell transformation. White related tyrosine kinase. Currently, tyrosine kinase inhibitors (TKI) (including imatinib, gleevet, etc.) have been widely used in CML treatment to prolong the survival of most CML patients and achieve cytogenetic or molecular biological remission. With the prolongation of the time of TKI clinical application, the problem of TKI resistance is becoming more and more obvious, actively exploring new Drug is an urgent problem.DBO (6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine) is a benzoxazine derivative. It has been proved that it can induce human umbilical vein endothelial cells to produce fine cell active oxygen substance (ROS) as a target protein of mammalian rapamycin target protein (m TOR) and induce the human umbilical vein endothelium. However, the antitumor effect of DBO has not been widely studied, and the mechanism of anti-tumor effect is not yet conclusive. The study of whether it causes autophagy and apoptosis in leukemia cells has not been reported. This study is based on the study of human K562 cells and the analysis of the proliferation of K562 cells by DBO and its induced cells. The effect of apoptosis and autophagy. The purpose of this study was to explore the effect of DBO on the proliferation, autophagy and apoptosis of human chronic myeloid leukemia K562 cells. Methods routine methods of resuscitation, generation of K562 cells, control group (DMSO), DBO treatment group, 24,48 and 72h were used to collect cells, and the expression of LC3 protein was detected by Western blot. Cell immunofluorescence test was used to detect the expression of LC3 spots; transmission electron microscopy was used to observe the cell autophagy by.CCK-8 colorimetric assay to detect the activity and proliferation of K562 cells. Cell cycle test was used to detect K562 cell mitosis by.Annexin V-FITC/PI double dye flow cytometry to detect cell apoptosis. Results of Western blot showed that K562 cells were Rima. The expression level of LC3- II protein increased and the expression level of p62 protein decreased. After treatment of 24,48 and 72 h by 50 mol/L DBO, the expression level of LC3- II and p62 protein was similar to that after the treatment of rapamycin, and the time dependent.K562 cells were treated with 10,25 and 50 mu mol/L DBO, and the protein expression level was increased. After the use of autophagy inhibitor 3-MA, the expression of LC3- II protein decreased and the expression of P62 protein increased. The cell immunofluorescence test showed that the expression of LC3 spots in the DBO treatment group increased after 72 h, compared with the control group. The results of transmission electron microscopy showed that in the cytoplasm of the DBO treatment group compared with the control group, the cytoplasm of the DBO treatment group was visible. More autophagosomes and autophagosomes, nuclei are irregular, and chromatin marginalization.CCK8 detection results show that DBO has a significant inhibitory effect on the proliferation of K562 cells, and the proliferation inhibition rate of time dose dependent.DBO by different concentrations (10 mu mol/L, 25 mu mol/L, 50 mu mol/L, 100 mu mol/L) is (0.68 + 0.05)% respectively (0.68 + 0.05)%. (2.76 + 0.35)%, (12.64 + 3.90)%, (22.58 + 2.41)%, F=67.389, P0.001, the difference between the treatment group and the control group was statistically significant, the proliferation inhibition rate of 48 h was (3.83 + 1.06)%, (6.23 + 1.27)%, (15.90 + 1.10)%, (15.90 + 1.10)%, F=, P0.001, and the difference between the treatment group and the control group was statistically significant; the proliferation inhibition rate of 72h was respectively, respectively. (8.78 + 1.28)%, (21.38 + 1.47)%, (32.11 + 2.01)%, (34.27 + 2.59)%, F=225.820, P0.001, the difference between the treatment group and the control group was statistically significant. The flow cytometry analysis showed that after DBO treatment 72 h, compared with the control group, the G2/M phase cells were increased compared with the control group, and the Chi 2=276.706, P0.001. flow cytometry analysis showed that compared with the control group, the analysis showed that the cells were compared with the control group. The apoptotic rate of K562 cells treated with DBO after 72 h treatment increased, 2=227.384 P0.001.. Conclusion DBO can significantly inhibit the growth of K562 cells, and is related to the occurrence of autophagy and apoptosis.
【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R733.72

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本文編號：2086702