本文關(guān)鍵詞： NSC67657 白血病細胞 單核系分化 ICAT Wnt/β-catenin信號通路　出處：《重慶醫(yī)科大學》2016年碩士論文　論文類型：學位論文

【摘要】：目的構(gòu)建新甾醇類藥物NSC6767誘導白血病細胞HL-60向單核系分化的細胞模型,檢測藥物對細胞的生物學行為的影響、探討其可能的分子機制。方法以10μmol/L NSC67657藥物處理HL-60細胞5 d構(gòu)建細胞分化模型,以DMSO溶劑處理HL-60細胞5 d為對照組;采用瑞氏染色、堿性α-丁酸萘酚酯酶(α-NBE)染色分析細胞的分化方向;采用流式細胞術(shù)分析細胞的分化程度、細胞周期分布、細胞凋亡的情況;采用乳酸脫氫酶(LD)檢測試劑盒檢測藥物對細胞的毒性作用;采用RT-PCR和Western blot分析ICAT、β-catenin、T細胞因子4(TCF-4)的m RNA和蛋白在細胞分化前后的表達情況;采用Western blot和免疫熒光染色方法檢測細胞分化前后β-catenin,TCF-4在細胞內(nèi)定位情況。采用RT-PCR和Western blot分析Wnt信號通路下游靶基因c-myc、細胞周期蛋白D1(cyclin D1)、TCF-1 m RNA和蛋白在細胞分化前后的表達情況;結(jié)果瑞氏染色后觀察發(fā)現(xiàn):細胞在10μmol/L NSC67657作用5 d后細胞質(zhì)明顯增多,核仁消失,細胞核呈圓形或不規(guī)則形;α-NBE染色結(jié)果顯示:對照組胞質(zhì)無色,為陰性,實驗組胞質(zhì)中出現(xiàn)鮮紅色的顆粒沉淀,為陽性,并能被氟化鈉(Na F)抑制,抑制率50%。流式細胞術(shù)(FCM)結(jié)果顯示:10μmol/L NSC67657作用5 d后的實驗組CD14+細胞數(shù)為(94.37±2.84)%,高于對照組[(1.31±0.09)%],差異有統(tǒng)計學意義(P0.01);實驗組G1/G0期細胞所占比例為(76.46±2.83)%,高于對照組[(59.4±5.42)%],差異有統(tǒng)計學意義(P0.05);實驗組S期細胞所占比例為(18.76±0.98)%,低于對照組[(34.38±2.61)%],差異有統(tǒng)計學意義(P0.05);藥物處理前后的HL-60細胞早期和晚期凋亡均無明顯改變(均P0.05);LD檢測結(jié)果顯示,LD活性也并無明顯改變(P0.05)。RT-PCR、western blot結(jié)果顯示:藥物作用后HL-60細胞中ICAT表達水平上調(diào)(均P0.01);β-catenin表達水平下調(diào)(均P0.01);細胞核中β-catenin蛋白的水平下調(diào)(P0.01);TCF-4表達水平無明顯改變(均P0.05);細胞核中的TCF-4無明顯改變(P0.05);Wnt信號通路下游靶基因c-myc、cyclin D1、TCF-1的m RNA和蛋白表達水平均下調(diào)(均P0.05)。免疫熒光染色結(jié)果顯示:細胞核中的β-catenin蛋白減少,TCF-4無明顯改變。結(jié)論NSC67657可誘導白血病細胞HL-60向單核系分化,抑制細胞增殖,但不誘導細胞凋亡,且對細胞無毒副作用;在HL-60向單核系誘導分化的過程中,ICAT表達上調(diào),Wnt信號通路關(guān)鍵調(diào)節(jié)因子β-catenin及下游靶基因的表達下調(diào)。提示:ICAT可能通過抑制Wnt/β-catenin信號通路參與了NSC67657誘導白血病細胞HL-60向單核系分化的過程。
[Abstract]:Objective to establish a cell model of HL-60 differentiation into monocytes induced by NSC6767, a new sterol drug, and to investigate the effect of NSC6767 on the biological behavior of leukemia cells. Methods HL-60 cells were treated with 10 渭 mol/L NSC67657 for 5 days to construct a cell differentiation model. HL-60 cells were treated with DMSO solvent for 5 days as control group. The differentiation direction of the cells was analyzed by using Rayleigh staining and alkaline 偽 -NBEase staining. Cell differentiation, cell cycle distribution and apoptosis were analyzed by flow cytometry. Lactate dehydrogenase (LDD) assay kit was used to detect the cytotoxicity of the drug to cells. RT-PCR and Western blot were used to analyze ICAT-catenin. The expression of m RNA and protein of T cell factor 4 (TCF-4) before and after differentiation; 尾 -catenin was detected by Western blot and immunofluorescence staining before and after differentiation. The downstream target gene c-myc of Wnt signaling pathway was analyzed by RT-PCR and Western blot. The expression of cyclin D _ (1) cyclin D _ (1) RNA and TCF-1 m RNA and protein before and after cell differentiation; Results the results showed that after 5 days of treatment with 10 渭 mol/L NSC67657, the cytoplasm increased significantly, the nucleoli disappeared, and the nucleus was round or irregular. The results of 偽 -NBE staining showed that the cytoplasm of the control group was colorless and negative. In the experimental group, bright red granules were found in the cytoplasm, which were positive and could be inhibited by sodium fluoride. The results of flow cytometry showed that the number of CD14 cells in the experimental group treated with 10 渭 mol/L NSC67657 for 5 days was 94.37 鹵2.84). %. Higher than control group. [The percentage of G1 / G0 phase cells in the experimental group was 76.46 鹵2.83%, which was higher than that in the control group. [The percentage of S phase cells in the experimental group was 18.76 鹵0.98%, which was lower than that in the control group. [The difference was statistically significant (P 0.05). There were no significant changes in early and late apoptosis of HL-60 cells before and after drug treatment (all P 0.05). The results of LD test showed that the activity of LD did not change P0.05. RT-PCR. The results of western blot showed that the expression of ICAT in HL-60 cells was up-regulated (all P 0.01). The expression of 尾 -catenin was down-regulated (P 0.01). The level of 尾 -catenin protein in the nucleus was down-regulated (P 0.01). There was no significant change in the expression of TCF-4 (P 0.05). There was no significant change in TCF-4 in the nucleus. The downstream target gene of Wnt signaling pathway, c-myc cyclin D1, was detected. The expression of m RNA and protein in TCF-1 were all down-regulated (P0.05). The results of immunofluorescence staining showed that the 尾 -catenin protein in the nucleus decreased. Conclusion NSC67657 can induce the differentiation of leukemic cells into monocytes and inhibit the proliferation of leukemia cells, but it does not induce apoptosis and has no toxic and side effects on leukemic cells. The expression of HL-60 was up-regulated during its differentiation into monocytes. The expression of 尾 -catenin and downstream target gene in Wnt signaling pathway was down-regulated. ICAT may be involved in the differentiation of HL-60 into monocytes induced by NSC67657 by inhibiting Wnt- 尾 -catenin signaling pathway.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R733.7

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相關(guān)期刊論文 前1條

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本文編號：1484962