3-溴丙酮酸诱导鼻咽癌细胞死亡的作用及其机制

发布时间：2018-06-25 03:54

本文选题：鼻咽癌 + 3-溴丙酮酸　；参考：《蚌埠医学院》2016年硕士论文

【摘要】：目的:1.3-溴丙酮酸(3-Bromopyruvate,3-BrPA)抑制人鼻咽癌细胞HNE1和CNE-2Z细胞的增殖和诱导死亡的作用。2.3-BrPA作用于人鼻咽癌细胞是否通过活性氧(ROS)水平的变化引起细胞程序性坏死。方法:1.人鼻咽癌细胞HNE1及CNE-2Z经过药物处理后采用MTT法检测细胞存活率;集落克隆法检测药物处理后细胞集落形成情况;不同浓度的3-BrPA处理人鼻咽癌细胞HNE1及CNE-2Z 5h,用腺嘌呤核苷三磷酸(ATP)水平检测试剂盒检测。2.PI单染法用流式细胞仪检测3-BrPA(0,80,160,320μM)作用于人鼻咽癌细胞HNE1及CNE-2Z细胞死亡率。线粒体膜电位检测试剂盒(JC-1)检测3-BrPA(0,40,80,160μM)作用于人鼻咽癌细胞HNE1及CNE-2Z 24 h后的细胞线粒体膜电位变化。3.用Caspase抑制剂z-VAD-fmk(20μM)或RIPK1抑制剂necrostatin-1(Nec-1,20μM)预处理人鼻咽癌细胞HNE1及CNE-2Z 1h。预处理后,3-BrPA(160μM或320μM)联合使用z-VAD-fmk(20μM)或Nec-1(20μM)。采用MTT法检测存活率,来明确两株细胞死亡形式;最后将HNE1和CNE-2Z用3-BrPA(160μM或320μM)处理后,用电镜观察细胞形态和细胞器的变化,进一步验证两株细胞HNE1和CNE-2Z的死亡形式。4.Western blot法检测药物处理人鼻咽癌细胞HNE1及CNE-2Z对相关蛋白表达水平的影响。5.人鼻咽癌细胞HNE1和CNE-2Z分别用3-BrPA(160μM或320μM)处理不同时间后,在细胞内装载超氧阴离子探针(DHE),采用流式细胞术检测细胞内活性氧(ROS)的含量变化情况;并用活性氧抑制剂N-乙酰半胱氨酸(N-acetylcysteine,NAC,5m M)预处理细胞1h,用NAC与3-BrPA共同作用于细胞,继而检测细胞内的ROS水平的变化。最后,3-BrPA联合NAC处理细胞,使用MTT法检测细胞存活率。6.在裸鼠体内实验中,建立鼻咽癌裸鼠移植瘤模型。检测移植瘤的生长曲线,HE染色指标观察3-BrPA在体内是否具有抑制肿瘤生长的效果。结果:1.3-BrPA对鼻咽癌细胞HNE1和CNE-2Z增殖的影响1.1采用MTT法检测,分析量效曲线可知在3-BrPA作用后人鼻咽癌细胞HNE1和CNE-2Z的增殖可以明显被抑制。并且随着3-BrPA作用于细胞的时间延长和浓度的逐渐增加,细胞增殖抑制作用明显增加。1.2检测3-BrPA对两株细胞HNE1和CNE-2Z增殖作用的影响,首先根据细胞存活率实验所得结果选用低于细胞IC50的药物浓度,观察集落形成数目。结果可以得出:低剂量浓度的3-BrPA对HNE1和CNE-2Z具有增殖抑制作用。1.3检测细胞内ATP含量,由实验结果显示3-BrPA(0,20,40,80,160,320μM)可以抑制鼻咽癌细胞内的ATP生成。2.3-BrPA诱导人鼻咽癌细胞死亡。2.2用不同浓度的3-BrPA作用于人鼻咽癌细胞HNE1和CNE-2Z,PI单染法检测结果显示:随着3-BrPA浓度增加,细胞死亡率增多。2.3 DAPI荧光染色法检测细胞核变化,结果显示随着3-BrPA浓度的增加细胞核呈现出浓缩致密,核裂现象逐渐增多。3.3-BrPA诱导线粒体膜电位发生变化。3.1应用荧光显微镜观察JC-1染色后,人鼻咽癌细胞线粒体膜电位变化,给药组同对照组的细胞相比较,可以观察到红色荧光逐渐向绿色荧光转变。这表明细胞内线粒体膜电位逐渐降低,细胞活性降低。3.2通过Western blotting法检测与有关蛋白Mcl-1、Bcl-2、Bax及IAPs蛋白家族的变化,结果显示:蛋白Mcl-1、Bcl-2以及IAPs蛋白家族CIAP1、CIAP2、XIAP表达减少,蛋白Bax的表达增多。4.ROS产生在3-BrPA诱导的鼻咽癌细胞死亡中的作用。4.1将两株细胞用3-BrPA处理,在显微下观察得荧光强度。可以观察到细胞内活性氧水平明显升高。4.2检测细胞内平均荧光强度采用流式细胞仪。同空白组相比,3-BrPA作用不同时间段后,可以观察到随着药物作用时间延长细胞内活性氧水平明显增高。NAC和3-BrPA联合用药组细胞内ROS水平没有明显升高。4.3细胞存活率检测实验,结果表明NAC(5m M)可以抑制3-BrPA诱导的细胞死亡。表明3-BrPA是细胞内ROS水平失衡而诱导的细胞死亡。5.3-BrPA诱导鼻咽癌细胞产生程序性坏死。5.1 Caspase抑制剂z-VAD-fmk(20μM)与3-BrPA联合使用后,HNE1和CNE-2Z的细胞存活率不受影响,而与RIPK1抑制剂Nec-1合用时,则发现细胞存活率增加。5.2 3-BrPA处理人鼻咽癌HNE1和CNE-2Z后电镜观察细胞,发现3-BrPA组细胞器膨胀,细胞呈空泡状,具有程序性坏死的特征。6.3-BrPA在裸鼠体内抗肿瘤效果6.1检测3-BrPA体内抑制肿瘤生长的作用,采用人鼻咽癌CNE-2Z异种移植瘤裸鼠模型。裸鼠肿瘤生长曲线表明:3-BrPA组的肿瘤生长曲线同空白组相比生长曲线平缓肿瘤体积较小,但效果略差于顺铂(DDP)组。苏木精-伊红染色法(HE染色)结果表明,3-BrPA给药组与空白组相比较起来,有大面积坏死区域存在。结论:1.3-BrPA对人鼻咽癌细胞HNE1和CNE-2Z的具有增殖抑制和诱导死亡的作用。2.3-BrPA诱导的鼻咽癌细胞死亡为程序性坏死,细胞死亡与细胞内ROS水平升高有关。
[Abstract]:Objective: 1.3- 3-Bromopyruvate (3-BrPA) inhibits the proliferation and induced death of HNE1 and CNE-2Z cells in human nasopharyngeal carcinoma cells and induces.2.3-BrPA to induce programmed cell necrosis in human nasopharyngeal carcinoma cells through the changes in reactive oxygen (ROS) levels. Methods: HNE1 and CNE-2Z in 1. nasopharyngeal carcinoma cells were treated with MTT. The cell viability was detected by the method of colony cloning, and the colony formation of the cells after treatment was detected by colony colony cloning. The 3-BrPA treatment of human nasopharyngeal carcinoma cells HNE1 and CNE-2Z 5h at different concentrations, the detection kit of adenine nucleoside three phosphoric acid (ATP) level detection kit and the.2.PI single staining method for the detection of 3-BrPA (0,80160320 uM) by flow cytometer in human nasopharyngeal carcinoma cell HNE1 Detection of CNE-2Z cell mortality. Mitochondrial membrane potential detection kit (JC-1) detection of 3-BrPA (0,40,80160 mu M) in human nasopharyngeal carcinoma cells HNE1 and CNE-2Z 24 h After pretreatment, 3-BrPA (160 mu M or 320 mu M) combined z-VAD-fmk (20 mu M) or Nec-1 (20 mu M). The survival rate was detected by MTT method to determine the death rate of two cells. Finally, the HNE1 and CNE-2Z were treated with 3-BrPA (160) or 320 micron. The changes of cell morphology and organelles were observed by electron microscopy, and two cells and the deaths were further verified. .4.Western blot method was used to detect the effect of drug treatment on human nasopharyngeal carcinoma cell HNE1 and CNE-2Z on the expression level of related proteins..5. human nasopharyngeal carcinoma cells HNE1 and CNE-2Z were treated with 3-BrPA (160 u M or 320 micron M) for different time, and the superoxide anion probe (DHE) was loaded in cells, and the content of intracellular reactive oxygen species (ROS) was detected by flow cytometry. The cell 1H was pretreated with the active oxygen inhibitor N- acetylcysteine (N-acetylcysteine, NAC, 5m M), and the NAC and 3-BrPA were used together to detect the changes in the level of ROS in the cells. Finally, 3-BrPA combined with NAC processing cells and used MTT method to test the cell survival rate in nude mice to establish nasopharyngeal carcinoma. The growth curve of the transplanted tumor was detected and the effect of 3-BrPA on the growth of tumor was observed by HE staining. Results: the effect of 1.3-BrPA on the proliferation of HNE1 and CNE-2Z in nasopharyngeal carcinoma cells 1.1 was detected by MTT method. The analysis of the volume effect curve showed that the proliferation of HNE1 and CNE-2Z in human nasopharyngeal carcinoma cells after 3-BrPA action was clear. The effect of 3-BrPA on the proliferation of HNE1 and CNE-2Z cells in two cells was significantly increased with the effect of.1.2 on the proliferation of HNE1 and CNE-2Z in two cells. First, the concentration of the cells below the cell IC50 was selected according to the results of the cell survival test, and the number of colony formation was observed. It can be concluded that low dose concentration of 3-BrPA has a proliferation inhibition effect on HNE1 and CNE-2Z,.1.3 detection of ATP content. The experimental results show that 3-BrPA (0,20,40,80160320 mu M) can inhibit the ATP generation.2.3-BrPA induced human nasopharyngeal carcinoma cell death in nasopharyngeal carcinoma cells, and the.2.2 3-BrPA acts on human nasopharyngeal cancer cells with different concentrations of 3-BrPA. The results of the single staining of CNE-2Z and PI showed that the cell death rate increased with the increase of 3-BrPA concentration and.2.3 DAPI fluorescence staining method to detect the nuclear change. The results showed that the nucleus appeared to be dense and dense with the increase of 3-BrPA concentration, and the phenomenon of nuclear fissure gradually increased by.3.3-BrPA induced mitochondrial membrane potential change of.3.1 applied fluorescence microscope view. After JC-1 staining, the mitochondrial membrane potential of human nasopharyngeal carcinoma cells was changed. Compared with the control group, the red fluorescence could be observed gradually to green fluorescence. This showed that the mitochondrial membrane potential decreased gradually and the cell activity decreased by the Western blotting method to detect the protein Mcl-1, Bcl-2, Bax and IAPs eggs by the Western blotting method. The changes in the white family showed that protein Mcl-1, Bcl-2 and IAPs protein family CIAP1, CIAP2, XIAP expression decreased, the expression of protein Bax increased.4.ROS in 3-BrPA induced nasopharyngeal carcinoma cell death.4.1 the two cells were treated with 3-BrPA, and the fluorescence intensity was observed under the microscope. The intracellular reactive oxygen water level could be observed. A flow cytometer was used to detect the average fluorescence intensity of.4.2 in the cells. Compared with the blank group, after the action of 3-BrPA in different time periods, it was observed that the level of intracellular reactive oxygen species increased obviously with the prolongation of drug action time, and the ROS level in the cells of.NAC and 3-BrPA was not significantly increased in the test of.4.3 cell survival rate. The results show that NAC (5m M) can inhibit the cell death induced by 3-BrPA. It is indicated that 3-BrPA is a cell death induced by the imbalance of ROS level in the cell, and.5.3-BrPA induces programmed necrosis of nasopharyngeal carcinoma cells to produce programmed necrosis of.5.1 Caspase inhibitor z-VAD-fmk (20 mu M), and the cell survival rate is not affected by the combined use of 3-BrPA. When EC-1 was used, the cell survival rate was found to be increased by.5.2 3-BrPA to treat HNE1 and CNE-2Z of human nasopharyngeal carcinoma, and the cells were observed by electron microscope after CNE-2Z. It was found that the organelle of the 3-BrPA group was expanded, the cell was vacuolated, and the characteristic of programmed necrosis was the anti tumor effect of.6.3-BrPA in nude mice 6.1 to detect the tumor growth in 3-BrPA body, and to use the CNE-2 in human nasopharyngeal carcinoma. The tumor growth curve in nude mice showed that the growth curve of the tumor growth curve in the 3-BrPA group was smaller than that in the blank group, but the effect was slightly worse in the group of Yu Shunbo (DDP). The results of the hematoxylin eosin staining (HE staining) showed that the 3-BrPA administration group was compared with the blank group, and there was a large area of necrotic area. Conclusion: the effect of 1.3-BrPA on the proliferation inhibition and induced death of HNE1 and CNE-2Z in human nasopharyngeal carcinoma cells is programmed with.2.3-BrPA induced death of nasopharyngeal carcinoma cells, and cell death is related to the increase of intracellular ROS level.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R739.63

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