四氟苯甲酰异亮氨酸二丁基锡酯（FPLDBT）诱导非小细胞肺癌A549细胞凋亡及其相关机制的研究

【作者】 胡尊丽；

【导师】 赵云峰；

【作者基本信息】 曲阜师范大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 研究背景和研究目的：肺癌是当今世界第一癌症杀手,在中国,其发病率与死亡率高居榜首,成为严重威胁人类健康和生命的一种疾病。按组织学类型可将肺癌分为小细胞肺癌(small cell lung cancer, SCLC)和非小细胞肺癌(non-small cell lung cancer, NSCLC)两大类。其中非小细胞肺癌(NSCLC)占肺癌的80%以上,而且就诊时多为Ⅲ～Ⅳ期,失去了手术机会,而在可手术的患者中,术后复发、转移率高达50%以上,且对放疗很不敏感。因此,全身化疗是治疗非小细胞肺癌的重要手段。目前所用化疗药物在化疗初期效果明显,可有效诱导肺癌细胞凋亡,但毒副作用严重且易产生耐药性,从而导致化疗失败。因此,寻找高效、毒副作用小、能克服耐药的新型化疗药物成为当前肺癌治疗亟待解决的主要问题之一。综上所述,本课题研究目的在于：以非小细胞肺癌A549细胞为研究对象,对合成的有机锡小分子化合物FPLDBT的体外抗肿瘤活性进行研究,进而探讨其对体外培养的非小细胞肺癌A549细胞的生长抑制作用及凋亡的分子机制,希望通过这些研究可以为治疗肺癌和开发新型肺癌化疗药物提供一定的理论依据,做出积极的贡献。研究方法：1.MTT法检测细胞存活率,对FPLDBT进行初步筛选。2.细胞凋亡情况的检测：—倒置相差显微镜观察细胞形态变化；—Hoechst 33258荧光染色,并结合荧光显微镜观察细胞核片段化；—吖啶橙(AO)荧光染色,结合荧光显微镜观察进一步检测细胞凋亡情况；—活性氧(ROS)水平的变化,用荧光分光光度计检测细胞内活性氧的含量；—用Nucleosome ELISA试剂盒检测细胞凋亡率。3.细胞周期影响的检测：—流式细胞技术检测FPLDBT对细胞周期的影响。4.FPLDBT对caspase-3,-4激活情况的检测,方法如下：—用caspase-3,-4酶活性检测试剂盒分析caspase-3,-4酶活性变化；—用caspases抑制剂检测caspase-3,-4对FPLDBT细胞生长的影响。研究结果：1.FPLDBT抗凋亡活性检测结果(MTT法检测细胞存活率结果)—FPLDBT对A549细胞存活率的影响：结果显示：在48小时内,FPLDBT显著降低了A549细胞的存活率,对A549有显著生长抑制作用,并呈现明显的剂量效应。2.FPLDBT诱导A549细胞凋亡的研究结果—倒置相差显微镜观察细胞形态变化：结果表明：0.8μM／L的FPLDBT处理A549细胞48小时后,倒置相差显微镜下观察发现,细胞皱缩,空泡化明显,产生凋亡小体,并从皿底脱落死亡,细胞呈现出典型的凋亡形态学特征；而0.1% DMSO溶剂对照组与对照组相比,细胞形态无明显变化。—Hoechst 33258染色结合荧光显微镜检测细胞核片段化结果：结果显示：0.8μM／L的FPLDBT处理A549细胞48小时后,荧光显微镜下观察发现,细胞核呈蓝色,出现核片段化现象,凋亡小体清晰可见,呈现典型的凋亡形态学特征；而DMSO溶剂对照组与对照组相比,细胞核呈均匀亮蓝色,形态无明显变化。—吖啶橙(AO)染色结合荧光显微镜检测染色质凝集结果：结果显示：0.8μM／L的FPLDBT处理A549细胞48小时后,荧光显微镜下观察发现,染色质凝集及边缘化,并有凋亡小体出现,呈现典型的凋亡形态学特征；而DMSO溶剂对照组与对照组相比,染色质无明显变化。—A549细胞内活性氧含量的测定：结果表明：0.8μM／L的FPLDBT处理A549细胞48小时后,结合荧光分光光度计检测其细胞内活性氧的含量发现,与对照组细胞相比,细胞内活性氧含量显著增加。—Nucleosome ELISA检测核小体水平变化结果：结果表明：0.8μM／L的FPLDBT处理A549细胞48小时后,Nucleosome ELISA试剂盒检测发现,与对照组细胞相比,细胞内核小体水平显著升高。3.FPLDBT对A549细胞周期的影响—流式细胞技术检测细胞周期分布结果：结果显示：通过流式细胞仪检测细胞周期分布发现,与对照组相比,0.8μM／L的FPLDBT作用48小时后可将绝大部分A549细胞周期阻滞在G1期,不能进入S期。4.FPLDBT诱导A549细胞凋亡的相关分子机制研究结果—caspases酶活性检测：结果表明：在细胞凋亡过程中,FPLDBT同时激活了caspase-3，-4,其中对caspase-4的激活程度强于caspase-3。—caspases抑制剂对FPLDBT诱导A549细胞凋亡的影响：加入caspases抑制剂后,凋亡ELISA试剂盒检测细胞凋亡率,发现：与FPLDBT处理的细胞相比,caspase-4 inhibitor(Z-LEVD-FMK)显著阻滞了FPLDBT诱导的凋亡,而caspase-3 inhibitor(Z-DEVD-FMK)虽然也抑制了FPLDBT诱导的凋亡,但程度较弱。研究结论：1.FPLDBT以浓度依赖的方式显著降低了A549细胞的存活率,抑制了A549细胞的增殖。2.FPLDBT可使细胞周期阻滞在G1期。3.FPLDBT可有效诱导A549细胞凋亡,此凋亡过程由ROS和caspases共同参与。4.根据caspases在FPLDBT诱导A549细胞的凋亡过程中所起的作用,我们推测此凋亡过程以内质网凋亡途径为主。更多还原

【Abstract】 Background and objective:Lung cancer is the most dangerous cancer throughout the world, and the incidence and death rate of lung cancer has ranked the first in China. According to histological type, lung cancer includes two main types:small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Therefore, it is particularly important for development and research of chemotherapeutic drugs.Chemotherapeutics is the most important treatment for lung adenocarcinoma because it is usually at the later stage when we find this kind of disease in the sufferers, at this moment, NSCLC becomes not suitable for surgery, and not sensitive to radiotherapy. At present, the frequently-used drugs for lung cancer chemotherapy can induce apoptosis of lung cancer cells, but strong toxicity side effect and drug resistance may cause failure of chemotherapy. Therefore, looking for the chemotherapeutic drugs which has high efficiency, minor toxicity, less side effect and ability to overcome multi-drug resistance is one of the urgent problems for researchers to be settled.In summary, A549 cells were selected as experimental cells to investigate in vitro screening of apoptosis-inducing drugs and molecular mechanisms of apoptosis. As well as the synthesis of organotin compounds FPLDBT anti-tumor activity in vitro study, and then, to explore the growth inhibition by FPLDBT and the molecular mechanisms of FPLDBT-induced apoptosis in A549 cells in vitro. We hoped that these studies could be in the treatment of lung cancer at the cellular level and the development of new chemotherapeutic agents for the treatment of lung cancer to provide a more complete theoretical basis, and make a positive contribution.Methods:1.MTT assay on cell viability, by which the FPLDBT was screened initially.2.Detection of cell apoptosis:-Observation of cell morphological changes by Phase Contrast Microscope.-Observation of nuclear fragmentation by Hoechst 33258 staining combined with Fluorescence Microscopy.-Observation of morphological changes of apoptosis by Acridine orange (AO) fluorescence staining combined with Fluorescence Microscopy.-Detection of the activity of ROS in A549, the content of ROS was determined by colorimetric assay.-Detection of apoptosis rates by Nucleosome ELISA assay kits.3.Detection of changes in cell cycle distribution:-Flow cytometry was used to detect the changes about the cell cycle of A549 after treated with FPLDBT. 4. Methods for determining whether caspase-3,-4 were activated by FPLDBT, as follows:-Analyzing caspase-3,-4 activities by caspase-3,-4 colorimetric assay kits.-Identification the influences of caspase-3,-4 on apoptosis induced by FPLDBT with caspase inhibitors.Results:1.The test results of anti-tumor activity of FPLDBT (the results of MTT assay on cells livability)-Influences of FPLDBT on viability of A549 cell: The results showed that:Within 48 hours, FPLDBT significantly reduced the survival rate of the A549 cell and significantly inhibited the growth of the A549 cell in a dosage dependent manner.2. Results of FPLDBT-induced apoptosis in A549 cells-Observation of morphological changes by Fluorescent inverted phase contrast microscope: The results showed that:After A549 cells were exposed to 0.8μM/L FPLDBT for 48 hours, cell morphological changes by Phase Contrast Microscope showed that cell shrinkage, vacuolization evident, apoptotic bodies occurred, and many cells detached from the dishes, showing a typical morphological characteristics of apoptosis; While treated with 0.1% DMSO, cell morphology had no obvious changes.-Results of detection of nuclear fragmentation by Hoechst 33258 staining combined with fluorescence microscopy: The results showed that:After A549 cells were exposed to 0.8μM/L FPLDBT for 48 hours, cell morphological changes by fluorescence microscopy revealed a uniform light blue nuclei, chromatin condensation, apoptotic bodies clearly visible, showing the typical morphological characteristics of apoptosis; While the DMSO group and the control group had no obvious change in cell nuclear morphology.-Results of detection of cell apoptosis by Acridine orange (AO) staining combined with fluorescence microscopy: The results showed that:After A549 cells were exposed to 0.8μM/L FPLDBT for 48 hours, cell morphological changes by fluorescence microscopy showed that pehromatin condensation and marginalization, emerging nuclei fragmentation, apoptotic bodies clearly visible, showing the typical morphological characteristics of apoptosis; While the DMSO group and the control group had no significant change in cell morphology.-The results of the activity test of ROS: The results showed that:When A549 cells were treated with 0.8μM/L FPLDBT for 48 hours, compared with the control group, the activity of ROS increased a lot.-Results of Nucleosome ELISA assay: The results showed that:When A549 cells were treated with 0.8μM/L FPLDBT for 48 hours, compared with the control group, FPLDBT could effectively induce apoptosis in A549 cells, and showed a dose-time-dependent manner.3.Influence of FPLDBT on the cell cycle of A549-Influence of FPLDBT on A549 cells cycle distribution: The results showed that:Flow cytometry analysis demonstrated that cells, after being treated with FPLDBT (0.8μM/L) for 48 hours, compared with the control group, most were blocked in G1 phase, could not enter S phase, in a time-dependent manner.4. Research findings of FPLDBT-induced apoptosis in A549 cells and its related molecular-Assay results of activities of caspases: The results showed that:In the apoptotic process, FPLDBT activated caspase-3 and-4, in which the activation of caspase-3 level was weaker than caspase-4.-Detection influences of caspases inhibitors on FPLDBT-induced apoptosis of A549 cells: Compared to 0.8μM/L FPLDBT treatment group, caspase-4 inhibitor (Z-LEVD-FMK) significantly reversed the FPLDBT-induced apoptosis in A549 cells. And caspase-3 inhibitor (Z-DEVD-FMK) reversed partially the FPLDBT-induced apoptosis.Conclusion:1. FPLDBT could significantly inhibit the proliferation of A549 cells in a concentration-dependent manner.2. FPLDBT induced A549 cells arrest at G1.3. FPLDBT could effectively induce A549 cells apoptosis in a dose-time-dependent manner. ROS and caspases might participate in this process of apoptosis.4. According to the effect of caspases in the process of apoptosis induced by FPLDBT in A549 cells, we speculated that the endoplasmic reticulumial pathway might play a major role in this process of apoptosis.更多还原