發(fā)布時間：2018-01-20 04:11

  本文關(guān)鍵詞： 鹽霉素 胃癌 Caspase-3 NF-κB 凋亡　出處：《延安大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:探討鹽霉素單獨(dú)及聯(lián)合17-AAG對人胃癌細(xì)胞株SGC-7901體外增殖的影響,旨在明確鹽霉素單獨(dú)及聯(lián)合17-AAG對人胃癌細(xì)胞體外增殖抑制作用及其機(jī)制。方法:以人胃癌SGC-7901細(xì)胞株為研究對象,應(yīng)用MTT比色法檢測鹽霉素單獨(dú)及聯(lián)合17-AAG對胃癌細(xì)胞的增殖抑制率;倒置相差顯微鏡觀察細(xì)胞形態(tài)變化;熒光顯微鏡觀察經(jīng)PI及AO染色后細(xì)胞凋亡形態(tài)變化;流式細(xì)胞術(shù)檢測細(xì)胞周期及凋亡率的變化;Western blotting檢測Caspase-3蛋白表達(dá)的變化;免疫細(xì)胞化學(xué)法檢測NF-κBp65、FAS-L蛋白表達(dá)的變化;采用SPSS22.0軟件進(jìn)行統(tǒng)計(jì)分析,所有分析數(shù)據(jù)均用x±s表示,所有均數(shù)間的比較用單因素方差分析,多個均數(shù)間的多重比較用LSD-t檢驗(yàn),以P0.05為差異具有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.MTT結(jié)果顯示鹽霉素在一定的濃度范圍(1、2、4、8、16、32μmol/L)內(nèi)分別作用24h、48h、72 h可以有效抑制胃癌SGC-7901細(xì)胞的增殖(P0.05或P0.01)。抑制效果呈劑量和(或)時間的依賴性。鹽霉素(4、8、16、32μmol/L)聯(lián)合17-AAG(0.625μmol/L)可以有效抑制胃癌細(xì)胞的增殖(P0.05),且兩藥聯(lián)合抑制效果顯著高于單獨(dú)用藥組(P0.05),提示鹽霉素可以增加17-AAG對胃癌細(xì)胞的敏感性。且聯(lián)合作用效果也呈濃度和時間依賴性。2.倒置相差顯微鏡及熒光顯微鏡觀察可見典型的細(xì)胞凋亡形態(tài)改變。3.流式細(xì)胞儀檢測細(xì)胞周期,結(jié)果顯示4、8、16μmol/L鹽霉素使胃癌細(xì)胞周期阻滯在S期,它們所占細(xì)胞周期的比例分別為26.90%、34.57%、41.83%,與對照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。8μmol/L鹽霉素與0.625μmol/L17-AAG單獨(dú)及聯(lián)合均導(dǎo)致細(xì)胞阻滯在S期,S期比例分別為34.57%、26.92%、40.10%顯著高于對照組的23.52%(P0.05),聯(lián)合用藥組與單獨(dú)用藥組比較差異也具有統(tǒng)計(jì)學(xué)意義(P0.05)。4.流式細(xì)胞儀檢測細(xì)胞凋亡,結(jié)果顯示4、8、16μmol/L鹽霉素使胃癌細(xì)胞周期阻滯在S期,它們所占細(xì)胞周期的比例分別為26.90%、34.57%、41.83%,與對照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.05)。8μmol/L鹽霉素與0.625μmol/L17-AAG單獨(dú)及聯(lián)合均導(dǎo)致細(xì)胞阻滯在S期,S期比例分別為34.57%、26.92%、40.10%顯著高于對照組的23.52%(P0.05),聯(lián)合用藥組與單獨(dú)用藥組比較差異也具有統(tǒng)計(jì)學(xué)意義(P0.05)。5.結(jié)果顯示:4、8、16μmol/L鹽霉素作用胃癌SGC-7901細(xì)胞48h后,細(xì)胞的凋亡率分別為10.52%、14.69%、20.46%,與對照組的3.42%比較,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。8μmol/L鹽霉素與0.625μmol/L17-AAG單獨(dú)及聯(lián)合用藥后細(xì)胞的凋亡率分別為14.69%、7.96%、26.87%,與對照組比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05),聯(lián)合用藥組與單獨(dú)用藥組比較差異也具有統(tǒng)計(jì)學(xué)意義(P0.05)。6.Western bloting檢測顯示鹽霉素和17-AAG單獨(dú)用藥均能顯著上調(diào)SGC-7901細(xì)胞中的Caspase-3的表達(dá),且兩藥聯(lián)合應(yīng)用時比單獨(dú)用藥更為明顯。7.免疫細(xì)胞化學(xué)法檢測顯示鹽霉素單獨(dú)及與17-AAG聯(lián)合均能顯著下調(diào)NF-κBp65的表達(dá),顯著上調(diào)Fas-L的表達(dá),且聯(lián)合作用蛋白表達(dá)變化較單獨(dú)用藥時明顯。結(jié)論:1.鹽霉素能有效抑制人胃癌細(xì)胞SGC-7901的體外增殖,其作用呈劑量和時間依賴性;2.鹽霉素與17-AAG聯(lián)合應(yīng)用可以抑制人胃癌細(xì)胞SGC-7901的體外增殖,其作用呈劑量和時間依賴性;3.鹽霉素單獨(dú)及與17-AAG聯(lián)合均可以誘導(dǎo)胃癌細(xì)胞SGC-7901的凋亡,其作用機(jī)制可能與上調(diào)胃癌細(xì)胞內(nèi)Caspase-3、FAS-L和下調(diào)NF-κBp65的表達(dá)有關(guān)。
[Abstract]:Objective: To investigate the effects of salinomycin alone and combined with 17-AAG on the proliferation of human gastric cancer cell line SGC-7901 in vitro, in order to clear the inhibitory effect and mechanism of salinomycin alone and combined with 17-AAG on the proliferation of human gastric cancer cells in vitro. Methods: human gastric cancer cell line SGC-7901 as the research object, should be detected by MTT inhibition rate by salt colorimetric method in separate and combined with 17-AAG on proliferation of gastric cancer cells; morphologic changes were observed under inverted microscope; fluorescence microscopy to observe the changes of apoptosis and morphology of PI and AO staining; detection of cell cycle and apoptosis rate by flow cytometry; changes of Western blotting expression of Caspase-3 protein was detected; detection of NF- kappa Bp65 immunocytochemical method and the alteration of FAS-L expression; SPSS22.0 software was used for statistical analysis, analysis of all data were expressed by X + s, compared with the number of all the single factor variance analysis, multiple mean The multiple comparison by LSD-t test, with P0.05 as the difference was statistically significant. Results: 1.MTT results showed that salinomycin in a certain range of concentrations (1,2,4,8,16,32 mol/L) in 24h 48h 72, respectively, h can effectively inhibit the proliferation of SGC-7901 (P0.05 or P0.01). The inhibitory effect was dose and (or time dependent). Salinomycin (4,8,16,32 mol/L) and 17-AAG (0.625 mol/L) can effectively inhibit the proliferation of gastric cancer cells (P0.05), and the two drugs combined with inhibitory effect was significantly higher than the single drug group (P0.05), suggesting that salinomycin can increase the sensitivity of 17-AAG on gastric cancer cells and the combined effect. It was time and concentration dependent.2. inverted microscope and fluorescence microscope observation showed typical apoptosis morphology change of.3. cell cycle by flow cytometry, the results showed that 4,8,16 mol/L salinomycin to gastric cancer cell cycle arrest In the S period, their proportion of cell cycle were 26.90%, 34.57%, 41.83%, there was statistically significant difference compared with control group (P0.05).8 mol/L Salinomycin and 0.625 mol/L17-AAG alone and in combination resulted in cell cycle arrest at S phase S phase ratio were 34.57%, 26.92%, 40.10% was significantly higher than that of control group of 23.52% (P0.05), difference between combined treatment group and single medication group was statistically significant (P0.05) apoptosis detection by flow cytometry showed that.4., 4,8,16 mol/L the salinomycin gastric cancer cell cycle arrest in S phase, the proportion of cell cycle were 26.90%, 34.57%, 41.83%, there are significant differences compared with the control group (P0.05).8 mol/L Salinomycin and 0.625 mol/L17-AAG alone and in combination resulted in cell cycle arrest at S phase S phase ratio were 34.57%, 26.92%, 40.10% higher than that of the control group 23.52% (P0.05), combination group Compared with the single drug group was also statistically significant difference (P0.05).5. showed that 4,8,16 mol/L salinomycin effect SGC-7901 gastric cancer cell 48h, cell apoptosis rates were 10.52%, 14.69%, 20.46%, compared with 3.42% in the control group, the difference was statistically significant (P0.05).8 mol/L of Salinomycin and 0.625 mol/L17-AAG alone and combination after the cell apoptosis rates were 14.69%, 7.96%, 26.87%, and the control group had significant difference (P0.05). The difference between the combined group and single drug group was also statistically significant (P0.05).6.Western bloting showed that Salinomycin and 17-AAG alone could up regulate the expression of SGC-7901 Caspase-3 in the cells, and the combination of the two drugs is more obvious than the sole use of detection.7. immunocytochemistry showed salinomycin alone and in combination with 17-AAG could significantly downregulate NF- kappa Bp65 table Up, up regulate the expression of Fas-L, and the joint effect of protein expression than either drug alone was 1.. Conclusion: the proliferation of salinomycin can inhibit the growth of human gastric cancer cells SGC-7901 in vitro. The effect was dose and time dependent proliferation; 2. salinomycin combined with 17-AAG can inhibit human gastric cancer cells SGC-7901 in vitro. The effect was dose and time dependent; 3. salinomycin alone and in combination with 17-AAG could induce apoptosis of gastric cancer cell SGC-7901 and its mechanism may be related to the upregulation of Caspase-3 in gastric cancer cells, FAS-L and expression of NF- kappa Bp65.

【學(xué)位授予單位】：延安大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 鄭朝旭;鄭榮壽;張思維;陳萬青;;中國2010年胃癌發(fā)病與死亡分析[J];中國腫瘤;2014年10期

相關(guān)博士學(xué)位論文 前1條

1 邱慧玲;腫瘤干細(xì)胞促卵巢癌淋巴管生成及轉(zhuǎn)移的作用及機(jī)制研究[D];第三軍醫(yī)大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 趙月;鹽霉素可以抑制乳腺癌細(xì)胞的增殖和侵襲并有效殺傷干細(xì)胞[D];華中科技大學(xué);2012年

，

本文編號：1446794