本文選題：鼻咽癌 + 3-溴丙酮酸��； 參考：《蚌埠醫(yī)學院》2016年碩士論文

【摘要】：目的:1.3-溴丙酮酸(3-Bromopyruvate,3-BrPA)抑制人鼻咽癌細胞HNE1和CNE-2Z細胞的增殖和誘導死亡的作用。2.3-BrPA作用于人鼻咽癌細胞是否通過活性氧(ROS)水平的變化引起細胞程序性壞死。方法:1.人鼻咽癌細胞HNE1及CNE-2Z經過藥物處理后采用MTT法檢測細胞存活率;集落克隆法檢測藥物處理后細胞集落形成情況;不同濃度的3-BrPA處理人鼻咽癌細胞HNE1及CNE-2Z 5h,用腺嘌呤核苷三磷酸(ATP)水平檢測試劑盒檢測。2.PI單染法用流式細胞儀檢測3-BrPA(0,80,160,320μM)作用于人鼻咽癌細胞HNE1及CNE-2Z細胞死亡率。線粒體膜電位檢測試劑盒(JC-1)檢測3-BrPA(0,40,80,160μM)作用于人鼻咽癌細胞HNE1及CNE-2Z 24 h后的細胞線粒體膜電位變化。3.用Caspase抑制劑z-VAD-fmk(20μM)或RIPK1抑制劑necrostatin-1(Nec-1,20μM)預處理人鼻咽癌細胞HNE1及CNE-2Z 1h。預處理后,3-BrPA(160μM或320μM)聯(lián)合使用z-VAD-fmk(20μM)或Nec-1(20μM)。采用MTT法檢測存活率,來明確兩株細胞死亡形式;最后將HNE1和CNE-2Z用3-BrPA(160μM或320μM)處理后,用電鏡觀察細胞形態(tài)和細胞器的變化,進一步驗證兩株細胞HNE1和CNE-2Z的死亡形式。4.Western blot法檢測藥物處理人鼻咽癌細胞HNE1及CNE-2Z對相關蛋白表達水平的影響。5.人鼻咽癌細胞HNE1和CNE-2Z分別用3-BrPA(160μM或320μM)處理不同時間后,在細胞內裝載超氧陰離子探針(DHE),采用流式細胞術檢測細胞內活性氧(ROS)的含量變化情況;并用活性氧抑制劑N-乙酰半胱氨酸(N-acetylcysteine,NAC,5m M)預處理細胞1h,用NAC與3-BrPA共同作用于細胞,繼而檢測細胞內的ROS水平的變化。最后,3-BrPA聯(lián)合NAC處理細胞,使用MTT法檢測細胞存活率。6.在裸鼠體內實驗中,建立鼻咽癌裸鼠移植瘤模型。檢測移植瘤的生長曲線,HE染色指標觀察3-BrPA在體內是否具有抑制腫瘤生長的效果。結果:1.3-BrPA對鼻咽癌細胞HNE1和CNE-2Z增殖的影響1.1采用MTT法檢測,分析量效曲線可知在3-BrPA作用后人鼻咽癌細胞HNE1和CNE-2Z的增殖可以明顯被抑制。并且隨著3-BrPA作用于細胞的時間延長和濃度的逐漸增加,細胞增殖抑制作用明顯增加。1.2檢測3-BrPA對兩株細胞HNE1和CNE-2Z增殖作用的影響,首先根據細胞存活率實驗所得結果選用低于細胞IC50的藥物濃度,觀察集落形成數目。結果可以得出:低劑量濃度的3-BrPA對HNE1和CNE-2Z具有增殖抑制作用。1.3檢測細胞內ATP含量,由實驗結果顯示3-BrPA(0,20,40,80,160,320μM)可以抑制鼻咽癌細胞內的ATP生成。2.3-BrPA誘導人鼻咽癌細胞死亡。2.2用不同濃度的3-BrPA作用于人鼻咽癌細胞HNE1和CNE-2Z,PI單染法檢測結果顯示:隨著3-BrPA濃度增加,細胞死亡率增多。2.3 DAPI熒光染色法檢測細胞核變化,結果顯示隨著3-BrPA濃度的增加細胞核呈現出濃縮致密,核裂現象逐漸增多。3.3-BrPA誘導線粒體膜電位發(fā)生變化。3.1應用熒光顯微鏡觀察JC-1染色后,人鼻咽癌細胞線粒體膜電位變化,給藥組同對照組的細胞相比較,可以觀察到紅色熒光逐漸向綠色熒光轉變。這表明細胞內線粒體膜電位逐漸降低,細胞活性降低。3.2通過Western blotting法檢測與有關蛋白Mcl-1、Bcl-2、Bax及IAPs蛋白家族的變化,結果顯示:蛋白Mcl-1、Bcl-2以及IAPs蛋白家族CIAP1、CIAP2、XIAP表達減少,蛋白Bax的表達增多。4.ROS產生在3-BrPA誘導的鼻咽癌細胞死亡中的作用。4.1將兩株細胞用3-BrPA處理,在顯微下觀察得熒光強度�？梢杂^察到細胞內活性氧水平明顯升高。4.2檢測細胞內平均熒光強度采用流式細胞儀。同空白組相比,3-BrPA作用不同時間段后,可以觀察到隨著藥物作用時間延長細胞內活性氧水平明顯增高。NAC和3-BrPA聯(lián)合用藥組細胞內ROS水平沒有明顯升高。4.3細胞存活率檢測實驗,結果表明NAC(5m M)可以抑制3-BrPA誘導的細胞死亡。表明3-BrPA是細胞內ROS水平失衡而誘導的細胞死亡。5.3-BrPA誘導鼻咽癌細胞產生程序性壞死。5.1 Caspase抑制劑z-VAD-fmk(20μM)與3-BrPA聯(lián)合使用后,HNE1和CNE-2Z的細胞存活率不受影響,而與RIPK1抑制劑Nec-1合用時,則發(fā)現細胞存活率增加。5.2 3-BrPA處理人鼻咽癌HNE1和CNE-2Z后電鏡觀察細胞,發(fā)現3-BrPA組細胞器膨脹,細胞呈空泡狀,具有程序性壞死的特征。6.3-BrPA在裸鼠體內抗腫瘤效果6.1檢測3-BrPA體內抑制腫瘤生長的作用,采用人鼻咽癌CNE-2Z異種移植瘤裸鼠模型。裸鼠腫瘤生長曲線表明:3-BrPA組的腫瘤生長曲線同空白組相比生長曲線平緩腫瘤體積較小,但效果略差于順鉑(DDP)組。蘇木精-伊紅染色法(HE染色)結果表明,3-BrPA給藥組與空白組相比較起來,有大面積壞死區(qū)域存在。結論:1.3-BrPA對人鼻咽癌細胞HNE1和CNE-2Z的具有增殖抑制和誘導死亡的作用。2.3-BrPA誘導的鼻咽癌細胞死亡為程序性壞死,細胞死亡與細胞內ROS水平升高有關。
[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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R739.63

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