【作者】 刘鑫；

【导师】 单保恩； 艾军；

【作者基本信息】 河北医科大学 ， 临床检验诊断学， 2009， 硕士

【摘要】 目的:研究北豆根总碱对宫颈癌细胞Hela凋亡、细胞周期及侵袭性的影响,探讨北豆根总碱抗肿瘤作用机制,为抗肿瘤新药的研究和开发提供科学依据。方法:1采用四甲基偶氮唑蓝法(MTT)检测不同浓度北豆根总碱(3.125、6.25、12.5、25、50μg/ml)处理不同时间(24、48和72小时)对Hela细胞增殖反应的抑制作用。2采用流式细胞技术(flow cytometry,FCM)检测不同浓度北豆根总碱(0、4、16、40μg/ml)作用48小时,对Hela细胞凋亡及周期变化的影响。3瑞氏-姬姆萨染色后显微镜观察北豆根总碱(0、40μg/ml)作用24小时后Hela细胞形态学变化。4采用Transwell小室法检测北豆根总碱(0、40μg/ml)作用24小时后对Hela细胞侵袭性的影响。5采用免疫印迹(Western blot)方法检测不同浓度北豆根总碱(0、4、16、40μg/ml)作用24小时后,Hela细胞中磷酸化ERK(p-ERK)、ERK、C-myc、CyclinD1的表达变化。6采用逆转录-聚合酶链反应(revers transcription PCR,RT-PCR)半定量检测北豆根总碱(0、4、16、40μg/ml)作用24小时,Hela细胞抗凋亡基因bcl-2、促凋亡基因bax、基质金属蛋白酶-9(MMP-9)的表达变化。7应用激光共聚焦显微镜(laser scanning microscope,LSM)观察北豆根总碱(0、40μg/ml)作用24小时后,Hela细胞内P-ERK、ERK、Bcl-2、Bax蛋白的表达变化。结果:1北豆根总碱对Hela细胞增殖有较强的抑制作用,随着北豆根总碱浓度及作用时间的增加,对细胞的抑制率升高。其中50μg/ml北豆根总碱作用72小时对细胞抑制率最高(85.37%);与对照组相比,不同浓度北豆根总碱对细胞增殖的抑制率均有显著性差异(p<0.05)。2北豆根总碱能明显诱导Hela细胞发生凋亡和细胞周期阻滞。随着北豆根总碱作用浓度的增大,细胞的凋亡率升高,G0/G1期细胞明显增多,S期细胞明显减少,对照组与试验组之间比较有显著性差异(p<0.05)。3北豆根总碱作用24小时后,Hela细胞形态呈现凋亡前期改变。4 Transwell小室侵袭实验结果显示,经北豆根总碱(0,40μg/ml)作用24小时后,穿膜细胞数较对照组显著减少,存在显著差异(p<0.05)。5 Western blot分析结果显示,经4、16、40μg/ml北豆根总碱处理24小时后,Hela细胞中磷酸化ERK、C-myc、CyclinD1表达水平与对照组细胞比较均有所下降,ERK表达无显著性变化。6半定量RT-PCR检测结果显示,经不同浓度北豆根总碱处理后,Hela细胞抗凋亡基因bcl-2、基质金属蛋白酶-9 MMP-9 mRNA表达水平逐渐降低,促凋亡基因bax mRNA表达水平逐渐增高,与对照组比较有显著性差异(p<0.05),该现象随着浓度北豆根总碱浓度的升高而更加明显,呈明显的浓度依赖性。7 LSM荧光图像分析结果显示,北豆根总碱(40μg/ml)作用24小时后,Hela细胞内抗Bax蛋白抗染色的荧光强度较对照组明显增加,抗P-ERK、Bal-2抗体荧光强度较对照组明显降低,ERK抗体荧光强度较对照组无明显变化。结论:1北豆根总碱在一定浓度范围内,能抑制宫颈癌细胞Hela的增殖,具有明显的量效关系。2北豆根总碱可通过降低Hela细胞中磷酸化ERK(P-ERK)水平,抑制Hela细胞ERK1/2信号转导通路。3北豆根总碱通过抑制Hela细胞ERK1/2信号转导通路,减弱bcl-2,MMP-9 mRNA表达,增强bax mRNA表达,减弱Hela细胞中C-myc、Cyclin D1蛋白的表达,进而促进Hela细胞凋亡,抑制Hela细胞侵袭力,使细胞阻滞于G0/G1期。更多还原

【Abstract】 Objective: To study the effects of Total Alkaloid of Menispermum （TAM） on apoptosis, cell cycle and invasive ablity to the Hela cell. To investigate the antitumor mechanisms of TAM. To produce the scientific evidence for developing and manufacturing new antitumor drugs.Methods: 1 The inhibitory effect on cell growth of Hela was measured by MTT assay in treated or untreated groups （3.125, 6.25, 12.5, 25, 50μg/ml TAM and control） for three different treatment times （24h, 48h and 72h）.2 Apoptosis and cell cycle were measured by FCM in four experimental groups （0, 4, 16, 40μg/ml TAM） for 48h.3 Adopting Wright and Giemse’s staining to observe the morphology of Hela cells which treated with 40μg/ml TAM.4 Using invasion experiment to detect the Hela cells’invasive abilities which treated with 40μg/ml TAM.5 The protein expressional levels of P-ERK, ERK, C-myc and Cyclin D1 in Hela cells untreated or treated with 4, 16, 40μg/ml TAM for 24h were measured by Western blot.6 Expression of anti-apoptotic gene bcl-2, apoptotic gene bax and MMP-9 in Hela cells of four experimental groups （0, 4, 16, 40μg/ml TAM for 24h）, were observed by revers transcription PCR （RT-PCR）.7 The protein expression of P-ERK, ERK, Bcl-2 and Bax in Hela cells treated with 40μg/ml TAM for 24h observed by laser scanning microscopes （LSM）.Results: 1 TAM could significantly inhibit the growth of Hela cells in vitro （p<0.05）. The growth inhibition was in dose-dependent. The greatest inhibit rate was 85.37% observed in the treatment group of Hela cells by 50μg/ml TAM for 72 hours.2 The results of FCM showed that TAM can induce apoptosis and cell cycle arrest of Hela cells. After treated by TAM for 48h, the apoptosis rate of Hela cells increased markely with a dose-dependent manner （p<0.05）. Meanwhile, after treated with TAM, the cell cycles of Hela cells were changed, the cells in G₀/G₁ phase were increased and cells in S phase were decreased.3 Wright and Giemsa’s staining revealed that the Hela cells treated by TAM had had proapoptotic morphology.4 The results of invasion experiment showed that the invasive ability of Hela cells treated with TAM had decreased remarkable （p<0.05）, compared with the contral group.5 The results of western blotting revealed that TAM had down- regulated the level of phopholated ERK, C-myc and CyclinD1 expression in Hela cells in treated group with 4, 16, 40μg/ml TAM for 24 hours, compared with the control group （untreated） （p<0.05）,but the level of ERK expression didn’t change significantly. 6 TAM inhibited the mRNA expression of the bcl-2 and MMP- 9, increased the mRNA expression of apoptotic gene bax in Hela cells treated by 4, 16, 40μg/ml periplocin for 24 hours compared with untreated cells （p<0.05）.7 LSM images show that after treated with 40μg/ml TAM, the fluorescence intensity of P-ERK, Bcl-2 were significantly weaker, and fluorescence intensity of Bax was more increased than that in control group. but fluorescence intensity of ERK was no significant change.Conclusion: 1 TAM could inhibit significantly proliferation of the human cervix cancer Hela cells in vitro in dose-dependent and time-dependent manner.2 In the Hela cells, TAM could repress the ERK1/2 signaling pathway through downregulating the expression of P-ERK.3 TAM could inhibit significantly the expression of bcl-2 ,MMP-9 mRNA, increase the expression of bax mRNA, affect the expression of C-myc and CyclinD1, accordingly, the treatment of TAM induced apoptosis , arrested the cell cycle in G₀/G₁ phase and debilitated invasion ability of Hela cells in vitro.更多还原