【作者】 潘效红；

【导师】 苗俊英；

【作者基本信息】 山东大学 ， 细胞生物学， 2008， 硕士

【摘要】 小分子化合物诱导人肺腺癌A549细胞凋亡机理研究研究目的近年来,肺癌死亡率居于各种恶性肿瘤之首位,其中肺腺癌最常见并且其发病率最高。由于肿瘤发生和生长的主要原因在于细胞凋亡受到抑制,因此,诱导肺腺癌细胞凋亡是治疗肺腺癌的重要措施,也是降低肺癌死亡率的有力手段之一。本文以人肺腺癌A549细胞系为例,利用化学遗传学这一新兴技术平台,筛选有效的诱导肺腺癌细胞凋亡的小分子化合物并研究其凋亡诱导分子机制,为进一步研究小分子凋亡诱导剂的特异性靶蛋白这一后续工作奠定基础,同时为开发新型的肺腺癌治疗药物提供理论依据。研究内容1.对小分子化合物进行初步筛选2.鉴定小分子化合物对A549细胞的凋亡诱导作用3.研究小分子化合物诱导A549细胞凋亡的分子机制研究方法1.MTT法检测细胞存活率,由此对小分子化合物进行初步筛选2.倒置相差显微镜观察细胞形态变化3.LDH(乳酸脱氢酶)检测细胞是否出现坏死4.吖啶橙染色结合激光扫描共聚焦显微镜观察,检测细胞核凝集5.TUNEL染色检测DNA片段化和凋亡率6.荧光探针DCHF检测ROS表达水平7.利用TMRM染色检测细胞线粒体膜电位变化[Falchi et al,2005]8.免疫荧光化学法检测膜整连蛋白β4的表达研究结果1苯并噁嗪酮衍生物对A549细胞的生长抑制作用1.1 MTT检测结果表明,在24h-48h内,5种苯并嗯嗪酮衍生物(1-5~#)均能抑制A549细胞的生长,且其抑制作用呈现时间和浓度依赖性。1.2倒置相差显微镜观察细胞形态发现:200μM苯并嗯嗪酮衍生物(1-5~#)分别处理A549细胞24h,细胞明显缩小,有的细胞变圆。2二茂铁基吡唑并哌嗪化合物对A549细胞的凋亡诱导作用2.1 MTT检测结果表明,在24h-48h内,6种二茂铁基吡唑并哌嗪化合物(8a-8f)均能显著降低A549细胞的存活率并且其作用效果呈现时间和浓度依赖性。2.2倒置相差显微镜观察细胞形态发现:8d-8f各20μM分别处理A549细胞24h-48h,细胞呈现典型的凋亡形态学特征。2.3吖啶橙染色结合激光扫描共聚焦显微镜检测细胞核片段化,发现:20μM8d-8f分别处理A549细胞48h时,细胞核固缩、染色质凝集、凋亡小体清晰可见。2.4 LDH检测发现:8d-8f各20μM分别处理A549细胞48h时,与对照组细胞相比,培养液中LDH酶活浓度未发生显著性变化(P＞0.05)。2.5 TUNEL染色发现,8d-8f各20μM分别处理A549细胞48h,TUNEL染色阳性率显著升高(P＜0.01)。2.6使用荧光探针DCHF检测细胞内ROS水平的变化,结果发现,8d-8f各20μM分别处理A549细胞48h时,细胞内的ROS水平显著升高(P＜0.01)。2.7利用TMRM染色检测细胞线粒体膜电位的变化,结果显示,8d-8f各20μM分别处理A549细胞48h时,细胞内的线粒体膜电位显著升高(P＜0.01)。2.8免疫细胞化学检测发现,8d-8f各20μM分别处理A549细胞48h时,膜整连蛋白β4的水平显著升高(P＜0.01)。结论1.小分子化合物初步筛选结果表明,5种苯并噁嗪酮衍生物对A549细胞生长均有抑制作用,其中2~#、3~#和5~#化合物的抑制作用比较明显。2.MTT初步筛选结果显示,6种二茂铁基吡唑并哌嗪化合物(8a-8f)对A549细胞增殖均有抑制作用,但其增殖抑制能力不同,其中8d、8e、8f三种小分子化合物增殖抑制能力较强,因此选取该三种小分子继续研究其对A549细胞的作用及其分子机制。3.8d、8e、8f三种小分子化合物20μM均能诱导A549细胞凋亡,而该凋亡诱导作用可能是通过提高ROS水平和影响线粒体膜电位来实现的。4.在8d、8e、8f三种小分子化合物诱导A549细胞凋亡的过程中,膜整连蛋白β4的表达水平升高,说明膜整连蛋白β4可能是该类小分子化合物诱导A549细胞凋亡途径中的一个重要因子。更多还原

【Abstract】 INVESTIGATION OF SMALL MOLECULE-INDUCED APOPTOSIS AND THE MOLECULAR MECHANISMS IN A549 HUMAN LUNG CANCER CELLSIn recent years,lung cancer is the leading category of cancer threatening the health of the people in many countries,lung cancer mortality stands the first within all kinds of cancer death.Moreover,among all types of lung cancer,adenocarcinoma occures most commonly and its incident rate is the highest.More and more studies evident that cell apoptosis inhibition is the major cause of tumorigenesis and progresss.Therefore,induction of apoptosis by drugs in lung adenocarcinoma cells is an indispensable strategy for lung adenocarcinoma therapy,which would decrease lung cancer mortality.In this article,we aimed to screen small molecules to effectively promote lung adenocarcinoma A549 cell apoptosis by using Chemical Genetics,the novel technology platform.Besides,we also aimed to investigate the mechanisms by which the small compounds function.This study would lay the foundation for us to insight into the proteins targeted by the small molecules,and would provide a theoretical basis for developing anti-lung adenocarcinoma agents.STUDY CONTENTS:1 Screening the small molecules initially.2 Identification of apoptosis induction in A549 cells by small molecules.3 Study the mechanisms of the small molecules inducing apoptosis in A549 cellsMETHODS:1.MTT assay for cell viability,by which small molecules were screened initially.2.The morphological changes were observed under phase contrast microscope.3.LDH assay to determine whether the cells treated by the small molecules undergo necrosis.4.DNA nuclear fragmentation was analyzed by acridine orange staining.5.The TdT-mediated dUTP nick end labeling technique was used to detect in situ nuclear DNA fragmentation and count apoptosis ratio6.The fluorescence probe,DCHF was used to analyze ROS level.7.The change of the mitochondrial membrane potential was measured by the TMRM staining as described previously[Falchi et al,2005]8.The expression of integrinβ4 was analysised by immunofluorescence assay.RESULTS:1.The effects of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives on A549 lung cancer cell growth1.1 The results of MTT assay showed that 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives(1-5~#)could inhibit A549 lung cancer cell growth within 24-48 h,and in a time and dose dependent manner.1.2 Cell morphological changes observed under Phase Contrast Microscope:When cells were exposed to 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives(1-5~#) 200μM for 24h,morphological changes were found,including cell shrinkage, round and cell viability decreased.2.The effects of 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a] pyrazin-4(5H)-one derivatives on A549 lung cancer cell growth2.1 The results of MTT assay showed that all kinds of six 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a] pyrazin-4(5H)-one derivatives(8a-8f)could decrease cell viability markedly within 24-48 h,and in a time and dose dependent manner.2.2 Cell morphological changes observed under Phase Contrast Microscope:When cells were exposed to 8d-8f 20μM for 24-48h,morphological changes which exhibiting the classical characteristics of apoptosis occurred.2.3 Nuclear fragmentation assay by AO staining in conjunction with laser scanning confocal microscopy(LSCM)showed that when incubation with 8d-8f 20μM for 48 h,many cells showed red-orange nuclear fragments and apoptotic bodies.2.4 LDH assay showed that when the cells were incubated with 8d-8f 20μM for 48 h,there was no significant difference(p＞0.05)in LDH release between the control group and the test group.2.5 TUNEL assay showed that the 8d-8f 20μM and 48h treatment group showed an overwhelming majority of apoptotic A549 cells(P＜0.01).2.6 When A549 cells were treated with 8d-8f 20μM for 48h,the intracellular ROS level was decreased obviously(P＜0.01).2.7 When A549 cells were treated with 8d-8f 20μM for 48h,the mitochondrial membrane potential level was decreased obviously(P＜0.01).2.8 The results ofβ4 protein expression detected by immuno-fluorescence assay combined with LSCM showed that when A549 cells were treated with 8d-8f 20μM for 48h,the relative level ofβ4 was up-regulated markedly(p＜0.01).CONCLUSION:1.Based on the initial screen of the small molecules,we concluded that all of the five kinds of small molecules could inhibit the proliferation of A549 human lung cancer cells,but their abilities in proliferation inhibition were quite different.2~#、3~# and 5~# were the most powerful.2.The results of MTT assay showed that all kinds of six 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a] pyrazin-4(5H)-one derivatives(8a-8f)could decrease cell viability markedly,but their abilities in proliferation inhibition were quite different.8d、8e and 8f were the most powerful,so our research was mainly focused on the effects of 8d-8f on the proliferation of A549 cells and the mechanisms by which the small compound functioned.3.8d、8e and 8f,20μM,could induce apoptosis in A549 human lung cancer cells. They might perform their functions by up-regulation of ROS level and effect the mitochondrial membrane potential level.4.Integrinβ4 level was up-regulation also,suggesting that integrinβ4 might be a key factor in the signal apoptosis.更多还原