【作者】 张云嵩；

【导师】 范士志；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 肺癌是人类最常见的恶性肿瘤之一,居恶性肿瘤死因的首位,其发病率及死亡率呈逐年上升的趋势。目前对肺癌的治疗方法有手术、放疗和化疗等,尽管这些治疗手段技术上均有了长足的进步,但是由于肺癌发生的分子机制及病理过程较为复杂,其5年生存率无明显提高,因此亟待探索新的肺癌治疗手段。光动力治疗（photodynamictherapy,PDT）是一种在有氧条件下,通过一定波长的激光激发靶细胞内光敏剂产生活性氧物质（reactive oxygen species,ROS）直接损伤肿瘤细胞、使微血管受损造成癌组织血流灌注减少的抗肿瘤新方法。但由于肿瘤细胞可能对其治疗过程中的氧化损伤及DNA损伤均具有一定的自身修复作用,影响了PDT对肿瘤的疗效。脱嘌呤/脱嘧啶核酸内切酶（apurinic apyrimidinic endonuclease,APE1）具有DNA损伤修复和氧化还原双功能,能通过调节生物体对氧化应激和缺氧的适应性,最大限度地减少细胞损伤。本研究以APE1为靶点,通过Ad5/F35-APE1 siRNA重组腺病毒,研究其体内对A549细胞感染效率和对APE1基因表达的“敲除”作用,及其体内及体外增强肺癌PDT敏感性的作用及机制。研究目的1.探讨APE1在非小细胞肺癌（non-small cell lung carcinomas,NSCLC）中的表达特点及其与患者预后的关系,为NSCLC以APE1靶点的基因治疗提供临床理论依据;2.探讨以APE1为靶点的基因治疗联合PDT在肺癌治疗中的临床应用前景。研究内容和方法1.APE1在NSCLC组织中表达及其临床意义:采用免疫组化方法检测NSCLC组织APE1蛋白表达特点,分析APE1表达高低与预后的关系,为进一步以APE1为靶点的基因治疗提供临床病理基础。2.Ad5/F35-APE1 siRNA重组腺病毒联合PDT对A549细胞杀伤效应的实验研究:采用流式细胞术检测Ad5/F35-APE1 siRNA重组腺病毒对A549细胞感染效率;Westernblot检测其对A549细胞APE1基因的沉默作用;采用[γ-³²P]ATP标记寡核苷酸法检测Ad5/F35-APE1 siRNA重组腺病毒对A549细胞AP内切酶活性的抑制作用;通过MTT来检测PDT及Ad5/F35-APE1 siRNA重组腺病毒联合PDT对A549细胞增殖抑制作用;流式细胞检测细胞凋亡;彗星实验检测DNA损伤;Western blot检测APE1蛋白及VEGF蛋白的表达。3.Ad5/F35-APE1 siRNA重组腺病毒联合PDT对A549细胞裸鼠移植瘤抑瘤作用的实验研究:建立肺腺癌A549细胞裸鼠移植瘤动物模型,观察Ad5/F35-APE1 siRNA重组腺病毒联合PDT对肺癌生长抑制的协同作用,TUNEL法检测肿瘤细胞细胞凋亡。研究结果1.APE1在NSCLC组织表达及其临床意义:正常肺组织及NSCLC组织均有APE1表达,但其表达的定位有所区别,正常肺组织以胞核表达为主,NSCLC组织以胞浆表达为主,两者之间差异性显著（P＜0.01）;通过Kaplan-Meier生存分析提示:APE1高低表达组之间生存时间有显著性差异,APE1低表达组中位生存时间为35±5.196个月,APE1高表达组中位生存期为26±0.761个月。2.Ad5/F35-APE1 siRNA重组腺病毒对A549细胞生物效应的实验研究:10 MOI的Ad5/F35-APE1 siRNA腺病毒对A549细胞的感染效率可达98%;Ad5/F35-APE1siRNA呈剂量依赖性地抑制A549细胞APE1蛋白表达,且能显著抑制A549细胞的AP内切酶活性。3.Ad5/F35-APE1 siRNA重组腺病毒增强A549细胞PDT治疗敏感性及机制的实验研究:Ad5/F35-APE1 siRNA联合PDT能显著增强A549细胞的增殖抑制作用;Ad5/F35-APE1 siRNA增加PDT诱导的A549细胞凋亡;碱性彗星分析结果显示Ad5/F35-APE1 siRNA抑制PDT后A549癌细胞DNA修复能力;PDT可诱导A549细胞APE1及VEGF蛋白表达增强,但通过Ad5/F35-APE1 siRNA可抑制A549细胞PDT诱导的APE1及VEGF蛋白的表达。4.Ad5/F35-APE1 siRNA重组腺病毒联合PDT对A549细胞裸鼠移植瘤抑瘤作用的实验研究:体内实验结果显示Ad5/F35-APE1 siRNA+PDT组肿瘤生长较单纯PDT组明显被抑制,单纯Ad5/F35-APE1 siRNA组肿瘤抑制率为34.85%,单纯PDT组肿瘤抑制率为52.19%,Ad5/F35-APE1 siRNA+PDT组肿瘤抑制率为93.97%。TUNEL检测肿瘤细胞凋亡情况,显示Ad5/F35-APE1 siRNA+PDT组肿瘤细胞凋亡率为（24.68±2.47）%,显著高于Ad5/F35-APE1 siRNA感染组（6.24±1.32）%、单独PDT组（12.12±1.76）%以及对照组（3.37±1.28）%（p＜0.01）。结论1.APE1在NSCLC中有胞浆异位表达的倾向,且APE1表达的高低与肺癌的预后有关。2.Ad5/F35-APE1 siRNA重组腺病毒容易感染肺腺癌A549细胞,且可特异性抑制A549细胞APE1蛋白表达,抑制A549细胞的AP内切酶活性。3.Ad5/F35-APE1 siRNA重组腺病毒能显著增强A549细胞PDT敏感性,主要是通过两种机制实现的,一方面通过抑制A549细胞PDT后的DNA损伤修复能力;另一方面通过抑制A549细胞对PDT所致氧化损伤的修复能力。4.PDT可诱导A549细胞APE1蛋白表达增强,但通过Ad5/F35-APE1 siRNA可抑制PDT诱导的APE1蛋白的表达。5.Ad5/F35-APE1 siRNA重组腺病毒联合PDT对A549细胞裸鼠移植瘤的抑瘤作用较单独PDT更为明显。更多还原

【Abstract】 Lung cancer is one of the most common malignancies,and is the leading cause of cancer death world wide.Moreover,the incidence rate and mortality of lung cancer tend to increase.The 3 main types of treatment for lung cancer are surgery,radiation therapy and chemotherapy.Although there is a great progress in treatment of lung cancer,5-year survival rates have not significantly improved,owing to complicated molecular mechanism involved in the carcinogenesis and development of lung cancer.Therefore,it is urgent to explore new approach for treatment of lung cancer.Photodynamic therapy（PDT） involves the application of a photosensitizer activated by visible light to generate reactive oxygen species（ROS）,which can result in anti-cancer effects through direct lethal direct lethal effects on tumor cells and vascular impairment which limit blood supply to tumor region. But DNA damage and oxidative damage self-repairing capability of tumor cells to PDT decrease sensitivity of tumor cells to PDT.The human apurinic/apyrimidinic endonuclease（APE1） play important role in DNA repair functions and response to oxidative stress and regulation of transcription factors.In this study,we investigated the effect of adenoviral vector Ad5/F35 carrying human APE1 siRNA on the sensitivity of PDT to human NSCLC in vivo and in vitro.Objective1.To research the expression of APE1 protein in NSCLC ceils,analyze the relationship between APE1 expression and prognosis of NSCLC patients,and determine that APE1 is an effective therapeutic target gene of NSCLC.2.To investigate the perspective of clinical application of combined treatment of adenoviral vector Ad5/F35-mediated APE1 siRNA with photodynamic therapy for lung cancer.Materials and Methods1.The expression of APE1 protein in NSCLC cells and its significance.The expression of APE1 in NSCLC patients was detected by immunohistochemical staining.To demonstrate that APE1 is an effective therapeutic target gene of NSCLC,the relationship between expression of APE1 and prognosis of NSCLC patients was determined.2.Efficacy of combination of Ad5/F35-APE1 siRNA with PDT therapy on A549 cells. The efficiency of infection of Ad5/F35-APE1 siRNA on A549 cells was assessed by flow cytometry and fluorescence.After transfection with Ad5/F35-APE1 siRNA,the protein levels of APE1 in A549 cells were evaluated by western blot The AP incision enzyme activity was evaluated by[γ-³²p]ATP-marked oligonucleotide assays.The effect of combination of PDT and Ad5/F35-APE1 siRNA transfection of A549 cell proliferation was assessed by MTT assay.DNA damage was investigated by comet analysis.The cell apoptosis was assessed by flow cytometry.The expression of APE1 and VEGF were detected by western blot3.The models of naked mice bearing transplantation tumor were established.Then,the sizes of the tumors were measured and the relevant growth curves were created.Afterwards, cell apoptosis was assessed by TUNEL assay.Results1.APE1 was detected in both lung tissues and NSCLC.While in the lung tissue,it was mainly observed in nuclear,whereas in NSCLC tussues it was observed in plasma.As shown by Kaplan-Meier survival analysis,the survival time of APE1-high expression group （median survival time:35±5.196 months） was longer than that of APE1-low group（median survival time:.26±0.761 months）.2.The infection efficiency of 10 MOI Ad5/F35-APE1 siRNA on A549 cells is 98%. Ad5/F35-APE1 siRNA transfection may inhibit APE1 protein expression in a dose-dependent manner.In the meantime,AP incision enzyme activity can be significantly inhibited.3.Application of PDT with Ad5/F35-APE1 siRNA transfection can significantly decrease the cell proliferation and increase the apoptosis rate of A549 cells.The results of comet assay showed that Ad5/F35-APE1 siRNA transfection can inhibit A549 cell DNA repairing ability after application of PDT.Ad5/F35-APE1 siRNA transfection can inhibit the increase in APE1 and VEGF protein induced by PDT.4.The results of in vivo study showed that tumor control rate in Ad5/F35-APE1 siRNA+PDT mice（93.97%） was higher than that of Ad5/F35-APE1 siRNA mice（34.85%） or PDT mice（52.19%）.The results of TUNEL assay showed that apoptosis rate of Ad5/F35-APE1 siRNA+PDT group（24.68±2.47%） was significantly higher than that of Ad5/F35-APE1 siRNA group（6.24±1.32%）,PDT group（12.12±1.76%） or control group （3.37±1.28%）.Conclusion1.Aberrant expression of APE1 in NSCLC may have a correlation with prognosis of lung cancer.APE1 seems to be a target for treating lung cancer,which may be of great clinical significance.2.Ad5/F35-APE1 siRNA transfection can infect A549 cells easily,which might specifically knockdown their APE1 expression.Also,it can markedly weaken the activity of incision enzymes.3.Ad5/F35-APE1 siRNA transfection significantly enhances sensitivity of A549 cells to PDT treatment,possibly through inhibition of DNA repairing capability after treatment with PDT,or via suppression of oxidation repairing of A549 cells in response to PDT.4.PDT might upregulate the expression of APE1 in A549 cells.However, Ad5/F35-APE1 siRNA might reverse this elevation.5.Combination of Ad5/F35-APE1 siRNA transfection with PDT can significantly inhibit the growth of transplantation tumors in naked mice.更多还原