塞来昔布诱导喉癌Hep-2细胞凋亡及细胞周期阻滞的实验研究

发布时间：2018-04-25 02:32

本文选题：环氧化酶-2 + 塞来昔布　；参考：《山东大学》2014年硕士论文

【摘要】：研究背景：喉癌在头颈部是一种比较常见的恶性肿瘤,近十年来其发病率有逐年升高的趋势。其传统治疗方法是手术切除,并根据患者情况辅助放疗或化疗。根据临床分期及病理分级,采取手术和(或)化疗及化疗联合放疗的综合治疗,被认为是喉癌治疗的理想方案。合理的化疗和(或)化疗联合放疗对于晚期及复发型喉癌病人总生存率、器官保留较传统手术都有提高。选择一种副作用小且有效的化疗药物在喉癌综合治疗中有重要作用。近十年来环氧化酶-2(COX-2)作为新的肿瘤治疗靶点成为肿瘤研究的热点,塞来昔布作为其特异性抑制剂,最初被美国FDA批准治疗疼痛,近年来发现其可以诱导包括消化道肿瘤在内的多种肿瘤细胞发生凋亡,抑制肿瘤生长,但塞来昔布在喉癌的临床应用和基础研究报道均较少。本研究,针对环氧化酶-2在人喉癌细胞系Hep-2细胞中的表达,及塞来昔布诱导Hep-2细胞凋亡和细胞周期阻滞进行初步研究,为探讨喉癌化疗敏感性提供理论依据。目的： 1.探讨COX-2在喉癌Hep-2细胞系中的表达及塞来昔布对COX-2表达的影响； 2.初步探讨塞来昔布诱导Hep-2细胞凋亡及可能的机制。 3.初步探讨塞来昔布诱导Hep-2细胞周期阻滞及可能的机制。方法：以不同浓度的塞来昔布处理喉癌Hep-2细胞,以四甲基偶氮唑盐(MTT)法检测对细胞增殖活力的影响；Hoechst33342对细胞进行染色,荧光显微镜下观察细胞核的形态改变；Annexin V/PI双染以流式细胞仪检测细胞的凋亡率；透射电镜观察塞来昔布处理后的细胞超微结构改变；JC-1染色,荧光显微镜下观察细胞线粒体膜电位的改变；Western blot检测：Bcl-2、Bax及COX-2的表达,细胞周期蛋白依赖性激酶抑制因子(cyclin-dependent-kinaseinhibitor;CKI) P16IM4a、P21waf/cip1、P27KIP的表达,以及分析Caspase-3、PARP裂解和AIF从线粒体释放到细胞核。结果： 1.COX-2在喉癌Hep-2细胞系中存在表达且其表达受塞来昔布抑制。 2.MTT结果显示塞来昔布在10-100μmol/L浓度范围可抑制Hep-2细胞的增殖活力,并呈时间和浓度依赖性。Hoechst33342染色及电镜结果可见镜下典型凋亡的形态改变；流式细胞仪结果显示Hep-2细胞凋亡率随塞来昔布浓度增加而升高。 3.流式细胞仪分析细胞周期分布可见细胞周期阻滞于G0/G,期。 4.JC-1染色可知,随着塞来昔布处理时间的延长,线粒体膜电位逐渐下降。 5. western blot结果见：随着塞来昔布浓度的升高,Bax/Bcl-2蛋白比值升高,P16INK4a、P27KIP表达增加,P21waf/cip1表达无明显改变；随着塞来昔布处理时间的延长,caspase-3蛋白、PARP蛋白逐渐出现裂解片断,AIF蛋白逐渐从线粒体释放移位到细胞核。结论：塞来昔布抑制肿瘤细胞增殖活性机制可能与其诱导细胞凋亡和细胞周期阻滞有关。塞来昔布抗肿瘤活性可能与COX-2途径有部分联系,是一种有前景的喉癌化疗药物。
[Abstract]:Background: laryngeal carcinoma is a common malignant tumor in head and neck. The traditional treatment is surgical resection and adjuvant radiotherapy or chemotherapy according to the patient's condition. According to the clinical stage and pathological grade, it is considered to be an ideal treatment for laryngeal cancer to take the combined treatment of surgery and / or chemotherapy and chemotherapy combined with radiotherapy. Reasonable chemotherapy and / or chemotherapy combined with radiotherapy can improve the overall survival rate and organ preservation in patients with advanced and recurrent laryngeal cancer. It is important to select a chemotherapeutic agent with small side effects in the treatment of laryngeal cancer. In recent ten years, cyclooxygenase-2 (COX-2) as a new target of tumor therapy has become a hot topic in cancer research. Celecoxib, as a specific inhibitor of cyclooxygenase-2, was initially approved by the United States FDA for the treatment of pain. In recent years, it has been found that celecoxib can induce apoptosis of many kinds of tumor cells, including digestive tract tumors, and inhibit tumor growth. However, there are few reports on the clinical application and basic research of celecoxib in laryngeal carcinoma. In this study, the expression of cyclooxygenase-2 in human laryngeal carcinoma cell line Hep-2, celecoxib induced apoptosis and cell cycle arrest of Hep-2 cells were studied. Objective: 1. To investigate the expression of COX-2 in Hep-2 cell line of laryngeal carcinoma and the effect of celecoxib on COX-2 expression. 2. To explore the possible mechanism of celecoxib induced apoptosis in Hep-2 cells. 3. To explore the mechanism of cell cycle arrest induced by celecoxib in Hep-2 cells. Methods: Laryngeal cancer Hep-2 cells were treated with celecoxib at different concentrations. Nuclear morphological changes were observed under fluorescence microscope Annexin V/PI double staining was used to detect cell apoptosis rate by flow cytometry, and ultrastructural changes of cells treated with celecoxib were observed by transmission electron microscope (TEM), and JC-1 staining was observed after celecoxib treatment. The changes of mitochondrial membrane potential were observed under fluorescence microscope. The expression of Bcl-2P Bax and COX-2 was detected by blot, and the expression of cyclin -dependent kinase inhibitor (CKI) P16IM4acip1wafP21waf1 / p27KIP was analyzed. The cleavage of Caspase-3PARP and the release of AIF from mitochondria into nucleus were analyzed. Results: 1.COX-2 was expressed in Hep-2 cell line of laryngeal carcinoma and its expression was inhibited by celecoxib. The results of 2.MTT showed that celecoxib could inhibit the proliferation of Hep-2 cells in the concentration range of 10-100 渭 mol/L, and the morphological changes of apoptosis were observed in a time-and concentration-dependent manner. Flow cytometry showed that the apoptosis rate of Hep-2 cells increased with the increase of celecoxib concentration. 3. Flow cytometry analysis of cell cycle distribution showed that cell cycle arrest at G _ 0 / G, phase. 4.JC-1 staining showed that mitochondrial membrane potential decreased with the prolongation of celecoxib treatment time. 5. The results of western blot showed that with the increase of celecoxib concentration, the expression of P16INK4aP27KIP was increased with the increase of Bax-Bcl-2 protein ratio. With the prolongation of caspase-3 protein and PARP protein of celecoxib, the cleavage fragment of AIF protein was gradually released and translocated from mitochondria to nucleus. Conclusion: The mechanism of celecoxib inhibiting tumor cell proliferation may be related to apoptosis and cell cycle arrest induced by celecoxib. Celecoxib may be a promising chemotherapeutic agent for laryngeal cancer due to its partial association with COX-2 pathway.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.65

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