紫草素对急性早幼粒细胞白血病NB4细胞增殖凋亡的影响及相关机制的研究

发布时间：2018-04-19 14:58

本文选题：紫草素 + NB4细胞　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:研究紫草素对人急性早幼粒细胞白血病NB4细胞增殖与凋亡的影响及其相关作用机制。方法:1.以NB4细胞株作为研究对象,用梯度浓度的紫草素分别作用于NB4细胞不同时间后,CCK-8实验测定细胞增殖情况;2.以0.3μmol/L紫草素作用于NB4细胞,培养1 d后,通过流式细胞术检测细胞周期的改变和凋亡的改变;3.经Hochest 33342实验,通过荧光显微镜检测NB4细胞的胞核的形态学改变;4.Western blot检测细胞c-Myc,PARP,Caspase 3,Cleaved PARP,Cleaved Caspase 3,ERK,p-ERK,JNK,p-JNK,p38MAPK及p-p38MAPK蛋白表达情况;5.构建NB4细胞裸鼠移植瘤模型,通过描绘肿瘤生长曲线分析紫草素对裸鼠肿瘤发展情况的作用;6.通过免疫组化技术检测紫草素对抑制瘤组织中增殖指标c-Myc表达的影响。结果:1.NB4细胞经紫草素处理后,细胞活力受到抑制(P0.01),并且具有时间依赖性及浓度依赖性;2.流式细胞术检测到NB4细胞经紫草素处理后,G1期细胞比例上升(P0.01),G2期和S期细胞比例下降(P0.01);细胞凋亡率明显增加(P0.01);3.紫草素处理NB4后,经Hochest 33342着色,在荧光显微镜下发现细胞表现出核固缩,染色质聚集凝集,核破碎等现象;4.紫草素作用细胞1 d后,Western blot检测到凋亡相关蛋白Cleaved PARP,Cleaved Caspase 3表达明显增多(P0.01),p-JNK和p-p38MAPK蛋白水平明显升高(P0.01),p-ERK和c-Myc蛋白表达水平明显降低(P0.05);5.紫草素能够显著抑制移植瘤生长(P0.05);6.免疫组化结果显示,紫草素处理组增殖蛋白c-Myc表达与对照组相比显著降低(P0.05)。结论:紫草素可能通过调节MAPK通路和c-Myc表达水平来抑制NB4细胞增殖诱导其凋亡,并可抑制NB4细胞裸鼠移植瘤生长。
[Abstract]:Aim: to study the effect of porphyrin on proliferation and apoptosis of human acute promyelocytic leukemia (NB4) cells and its related mechanism.Method 1: 1.NB4 cell line was used as the research object. The proliferation of NB4 cells was measured by CCK-8 assay after treated with the gradient concentration of porphyrin for different time.NB4 cells were treated with 0.3 渭 mol/L porphyrin for 1 day. The cell cycle and apoptosis were detected by flow cytometry.The morphological changes of the nucleus of NB4 cells were detected by Hochest 33342 assay. 4. Western blot was used to detect the expression of c-Myccine paspase 3 and Cleaved PARPASE p38 MAPK and p-p38MAPK protein in NB4 cells.The model of transplanted tumor in nude mice with NB4 cells was constructed, and the effect of porphyrin on tumor development in nude mice was analyzed by depicting tumor growth curve.The effect of Shikonin on the expression of c-Myc in tumor tissue was detected by immunohistochemical technique.Results: 1. The cell viability of Nb4 cells was inhibited after treated with porphyrin, and the cell viability was time-dependent and concentration-dependent.Flow cytometry (FCM) showed that the proportion of NB4 cells in G _ 1 phase increased after treatment with Shikonin, and the proportion of P0.01G _ 2 and S phase cells decreased (P _ (0.01)), and the apoptosis rate increased significantly (P _ (0.01)).After NB4 was treated with porphyrin, the cells were stained with Hochest 33342 and the cells showed nuclear pyknosis, chromatin aggregation and nuclear fragmentation under fluorescence microscope.One day after treatment with porphyrin, the expression of apoptosis-related protein Cleaved, p0.01, p-JNK and p-p38MAPK was significantly increased by Western blot. The expression level of P0.01PERK and c-Myc decreased significantly.Shikonin could significantly inhibit the growth of transplanted tumor.Immunohistochemical results showed that the expression of proliferating protein (c-Myc) in the treated group was significantly lower than that in the control group (P 0.05).Conclusion: Shikonin may inhibit the proliferation and apoptosis of NB4 cells by regulating the MAPK pathway and the expression of c-Myc, and inhibit the growth of NB4 xenografts in nude mice.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7

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