【作者】 张帅；

【导师】 李刚；

【作者基本信息】 山东大学 ， 临床医学（外科学·神经外科）（专业学位）， 2017， 博士

【摘要】 背景源自神经上皮的肿瘤统称为脑胶质细胞瘤(Glioma),是最常见的颅内原发恶性肿瘤,根据病理又可分为星形细胞瘤、髓母细胞瘤、多形胶母细胞瘤、室管膜瘤、少枝胶母细胞瘤等。在成人所有恶性肿瘤中占2%,在各类颅脑肿瘤类型中发病率最高。国外文献报道,胶质瘤约占颅内原发中枢神经系统肿瘤总数的50%左右,[1]占所有中枢神经系统恶性肿瘤的800%。根据我国统计数据,颅内肿瘤以胶质瘤为最多,占35.26%-60.960%(平均44.69%)。而胶质瘤中又有超过一半为高度恶性的胶质母细胞瘤。胶质瘤的综合发病年龄高峰有两个,主要在30-40岁之间,另外在10-20岁亦有一发病高峰。对于胶质瘤来说,性别对发生率差异应该是有意义的,男女发病率约为1.85:1。据报道,我国脑胶质瘤年发病率约十万分之三到十万分之六,年死亡人数超过3万人。胶质瘤根据WHO分级系统分为WHOⅠ-Ⅳ级,WHOⅠ-Ⅱ级为低级别胶质瘤,WHOⅢ-Ⅳ为高级别胶质瘤。在目前医疗水平下,胶质瘤的治疗预后仍然极差。除部分低级别胶质瘤,比如毛细胞星形细胞瘤,可实现手术治愈外,其他大部分胶质瘤在综合治疗后仍容易复发,预后极差。据文献报道,胶质母细胞瘤(WHOⅣ)平均生存时间仅为1年左右,其5年生存率仅为9.8%[2]胶质瘤多呈恶性,生长模式主要为浸润性、弥散性,具有难根治、易播散和易复发等特点。虽然针对胶质瘤的治疗,全世界已进行了各方面的研究和尝试,但预后仍然较差。尽管胶质瘤不同级别分型的恶性程度及预后有极大差距,但是目前针对胶质瘤的治疗主要都是以手术切除为主,术后放疗和化疗联合应用的综合治疗。《美国国家综合癌症网络(NCCN)指南》、《欧洲临床指南》、中国抗癌协会神经肿瘤专业委员会《中枢神经系统常见肿瘤诊疗纲要》和中华医学会《中国中枢神经系统胶质瘤诊断和治疗指南》对胶质瘤的治疗原则基本达成共识。[3,4,5]针对胶质母细胞瘤,口前国际公认的标准化治疗方案为Stupp方案(2005年)。Stupp方案强调首先在最大程度保护正常脑组织功能的条件下最大范围的彻底切除肿瘤,1月后给予放疗,并同期行替莫唑胺(TMZ)化疗,放疗结束后再给予6个周期的替莫唑胺化疗。[6]针对胶质瘤中的间变星形细胞瘤(WHOⅢ级),指南推荐仍可应用Stupp方案,或者行手术治疗后先单纯给予放疗,再给予化疗。针对低级别胶质瘤(WHOⅠ-Ⅱ级),手术治疗为公认的首选治疗,术后综合治疗未达成一致认同。普遍认为对于肿瘤全切且无高危因素患者可术后观察随访,对肿瘤残余或(和)有明确高危因素患者可酌情放化疗联合应用。Stupp方案报道10余年以来,胶质瘤尤其是高级别胶质瘤的治疗较前有了明显进展,但患者平均生存时间和预后仍不理想,其中位生存期仅为14.6个月。外科手术治疗是胶质瘤首选的,也是外科最基本的治疗方式,是综合治疗中最关键的前提。以上所提到的治疗共识均提到胶质瘤治疗的首选策略为外科手术。由于手术的目的在于降低死亡率、保护功能、提高生活质量,因此手术治疗的基本原则是在安全保护功能的基础上最大范围的切除肿瘤。循证医学的研究显示,手术切除程度与患者预后密切相关。[7]但是由于胶质瘤位于脑实质内,肿瘤呈浸润性生长,缺乏肉眼可分辨的清晰的分界区域,在临床中在保护脑功能的前提下很难做到完全的生物学全切。在胶质瘤的手术方式发展中,逐步经历了肉眼下全切肿瘤、显微镜下全切肿瘤和影像学全切肿瘤。目前发展出的手术辅助技术包括:(1)功能影像学术前定位应用;(2)神经导航术中应用;(3)术中唤醒技术术中应用;(4)术中B超、MRI等成像技术;(5)术中电生理检测的应用;(6)荧光显微镜及术中荧光造影的应用。以上技术的单独或联合应用,提高了术前定位的准确性,实现了术中规避重要功能区,纠正术中脑移位,显示残留肿瘤组织,提高了手术的安全性及扩大了切除范围。但是胶质瘤浸润性、弥漫性生长的特性决定了单纯手术无法做到生物学上的彻底全切,术后肿瘤残留和复发不可避免。因为如此局限性,在手术治疗基础上,又发展出放疗、化疗、免疫治疗、靶向治疗、基因治疗等辅助治疗。放疗是治疗胶质瘤的重要辅助手段,其治疗效果早已得到公认和肯定,许多研究表明术后放疗能够改善预后。[8,9,10]最初胶质瘤放射治疗为全脑放疗,脑放射损伤是常见的主要副作用。近年来,随着放疗技术的进步,逐步由全脑放疗转向为局部放疗,目的是在尽可能抑制肿瘤细胞的同时,减轻近期及远期正常脑组织损伤,改善预后,提高生活质量。目前又出现了立体定向放疗,三维适形放疗(3D-conformal radiation therapy,3D-CRT)、调强放疗技术(Intensity modulated radiation therapy,IMRT)等。Chan等的研究表明,新的放疗技术并未提高肿瘤的治疗效果。[11,12]目前精确放疗技术主要作用是减少正常脑组织的放射性损伤。由于胶质瘤细胞对放疗敏感性的差异性,放疗的有效性得不到保障。早期常规化疗药物对胶质瘤疗效改善有限。2005年新英格兰医学杂志发表了替莫唑胺(TMZ)同期放化疗的研究成果,在胶质瘤的放化疗史上具有划时代的意义。[6]在此基础上,Stupp方案已成为胶质母细胞瘤治疗的标准治疗方案。虽然替莫唑胺的出现使得胶质瘤化放疗有了长足进步,但其固有的毒副作用,以及不能透过血脑屏障、耐药性等缺陷,限制了其应用及疗效。口前兴起的靶向治疗研究主要针对于细胞增殖、血管生成相关的细胞表面受体和信号传导因子。主要包括:生长因子及受体抑制剂,细胞间信号传导抑制剂,抗血管生成药物等。但目前均无法单独对胶质瘤的预后产生重大影响,需要以上多种方案联合应用。胶质瘤发病率高,恶性程度高,预后较差,治疗效果不满意。人恶性胶质瘤对于目前所有的治疗方法都是一项严峻的挑战。因此,研究和开发新的有前途的抗胶质瘤药物成为迫切需求。[13]黄酮类(Flavonoids)化合物是存在于各种水果、蔬菜和植物饮料中的最重要和最大的一类植物化学物质,已知有8000多种不同的化合物。黄酮类化合物已被报道具有舒张血管、抗氧化、抗癌和抗致突变作用。[14]这些化学物质被认为针对多种慢性疾病,包括恶性肿瘤有显著的保护作用。这也解释了著名的癌症的风险减少和多吃含有黄酮类化合物的水果和蔬菜的关系。[15,16,17,18,19]查尔酮结构包括一个具有两个芳香环的开链的类黄酮,两个芳香环通过一个α-β不饱和碳基修饰的3-碳片段连接。他们是从植物中分离出来,被认为可以用来合成如黄酮和异黄酮等天然产物。[20]柚皮素查尔酮(柚苷配基苯乙烯基苯基甲酮)也属于黄酮类植物化学物质,存在于红色番茄的果皮中。[21]据我们所知,在公开发表的文献中还没有柚皮素查尔酮抗脑胶质瘤作用的报道。因此本研究的主要目的是通过评估柚皮素查尔酮对细胞凋亡、自噬和PI3K/Akt通路的作用,研究柚皮素查尔酮潜在的抗胶质瘤作用。目的研究的主要目的是探讨柚皮素查尔酮对U87MG胶质瘤细胞和异种移植小鼠模型的体外和体内抗胶质瘤作用。方法首先进行柚皮素查尔酮对U87MG胶质瘤细胞的体外抗肿瘤作用试验。将U87MG胶质瘤细胞中加入不同浓度(0,2.5,5,10,50,75和100 μM)柚皮素查尔酮,培养不同的时间(24,48,72h),分别用MTT比色法检测细胞增殖。将U87MG胶质瘤细胞中加入不同浓度(0,10,50和100 μM)柚皮素查尔酮培养48小时后,分别用吖啶橙/溴化乙锭和Hoechst33342染色后在荧光显微镜下评估细胞凋亡。将U87MG胶质瘤细胞中加入不同浓度(0,10,50和100μM)柚皮素查尔酮培养48小时后,在透射电子显微镜下观察细胞中自噬空泡的数量和大小。将U87MG胶质瘤细胞中加入不同浓度(0,10,75和100 μM)柚皮素查尔酮培养48小时后,Annexin V-FITC和PI染色处理后,使用流式细胞仪检测柚皮素查尔酮对U87MG人胶质瘤细胞的早期凋亡和晚期凋亡作用。将U87MG胶质瘤细胞首先于无血清培养基中进行饥饿处理4小时,然后加入不同浓度(0,10,50和100 μM)柚皮素查尔酮培养40分钟和48小时后用Western blot方法对柚皮素查尔酮对PI3K/Akt信号转导蛋白的作用进行评价。使用异种移植小鼠模型进行柚皮素查尔酮对U87MG胶质瘤细胞的体内抗肿瘤作用试验。将U87MG细胞皮下注射于裸鼠右后肢处建立异种移植小鼠模型。将建模成功小鼠分为5组,分别给予不同剂量柚皮素查尔酮注射(5,20,40 and 80 mg/kg)。24天后,处死小鼠,测量肿瘤的重量、长度和宽度,.计算肿瘤的体积。结果柚皮素查尔酮对U87MG细胞的增殖有抑制作用,其细胞毒性作用随着浓度的增加而增强,同时随着作用时间的增加而增强。也就是说,柚皮素查尔酮对U87MG细胞的细胞毒性作用同时具有剂量依赖性和时间依赖性。加入柚皮素查尔酮的U87MG细胞经过吖啶橙/溴化乙锭染色后,通过荧光显微镜可观察到细胞收缩与细胞凋亡体的形成。经hoechst 33342染色后,在荧光显微镜下可见染色质浓缩,DNA碎片,胞膜空泡。这些都是细胞凋亡的形态学表现特征。透射电子显微镜观察表明,柚皮素查尔酮能够诱导白噬空泡的形成,而且自噬空泡的数量和体积随柚皮素查尔酮剂量的增加而增加。流式细胞仪检测证明,柚皮素查尔酮能够诱导U87MG人胶质瘤细胞早期凋亡和晚期凋亡。柚皮素查尔酮同时能够抑制磷酸化和非磷酸化的PI3K和Akt蛋白,抑制NF-κB、Bcl-2,激活Caspase-3。体内实验结果显示,不同剂量柚皮素查尔酮干预组的肿瘤生长体积和重量均小于空白对照组。结论柚皮素查尔酮在U87MG人胶质母细胞瘤细胞和异种移植瘤小鼠模型中通过诱导凋亡、自噬作用和PI3K/Akt信号通路调控抗胶质瘤作用。更多还原

【Abstract】 BackgroundGlioma is the tumor Originated from nerve epithelium,is the most common primary intracranial malignant tumor.[1]Glioma can be divided into astrocytom,medulloblastoma,Glioblastoma multiforme(GBM),ependymoma,oligodendroglioma et.According to the pathological types.Glioma accounts for 2%of all malignant tumors in the adult,and the highest incidence in all types of braintumors.According to foreign literature,glioma accounts for about 50%of the primary central nervous system tumors,80%of all central nervous system malignancies.According to the statistics of our country,glioma is the most of the intracranial tumors,accounting for 35.26%-60.96%(average 44.69%).And more than half of gliomas are highly malignant glioblastoma.The age of onset has two peaks,mainly between 30-40 years old,and at the age of 10-20,there is also a peak.The incidence of gliomas and gender is significant,and the ratio of male to female is about 1.85:1.According to reports,the incidence of brain glioma in China is about 3/100000 to 6/100000,the number of deaths is more than 30 thousands one year.Glioma according to the WHO classification system is divided into WHOI-Ⅳ.The WHOI-Ⅱ grade is low grade glioma,and The WHOⅢ-Ⅳ grade is high grade glioma.At present medical level,the prognosis of the treatment of gliomas is still very poor.In addition to some low-grade gliomas,such as pilocytic astrocytoma,can be cured by surgery.Most of the other gliomas are still prone to relapse after comprehensive treatment,and the prognosis is very poor.According to the literature,the average survival time of glioblastoma(WHOIV)is only 1 year or so,the 5-years survival rate is only 9.8%.[2]Gliomas are malignant,and the growth pattern is mainly infiltration,dispersion,difficult to cure,easy to spread and easy to relapse.Although the world has carried out in all aspects of researches and tries on the treatment of glioma,but the prognosis is still poor.Although there is a great gap between the degree of malignancy and the prognosis of different grates of gliomas,the treatment of gliomas is mainly based on surgical resection,and the combination therapy of postoperative radiotherapy and chemotherapy."The National Comprehensive Cancer Network(NCCN)guidelines","European clinical guidelines"," common CNS tumor diagnosis and treatment program" and "China CNS glioma diagnosis and treatment guidelines"have reached a consensus on the principles of the treatment of gliomas.[3,4,5]For glioblastoma,the internationally recognized standard treatment program is Stupp program(2005).The Stupp program emphasizes that we must resect the tumor to the maximum rangeunder the condition of to protect the normal brain function in the greatest degree first,do the tadiotherapy and temozolomide(TMZ)chemotherapy at the same time after 1 month,and 6 cycles of chemotherapy after radiotherapy.[6]For the anaplastic astrocytoma(WHOⅢ),the guidelines recommend that you can still use the Stupp program,or give radiotherapy alone after surgery and then give chemotherapy.In view of low grade gliomas(WHOⅠ-Ⅱ),surgical treatment is generally accepted as the first choice and We have not reach a consensus about postoperative comprehensive treatment.It is generally believed that patients with total resection of the tumor and no high risk factors can be observed and followed up.The appropriate combined use of radiotherapy and chemotherapy can be given for patients with residual or(and)high risk factors.Stupp program is reported more than 10 years,the treatment of gliomas,especially high-grade gliomas,has been significantly more progress,but the average survival time and prognosis of patients are still not good enough,and the median survival time is only 14.6 months.Surgical is the first choice,the most basic,the most direct treatment of glioma,but also the most important prerequisite for comprehensive treatment.All of the above treatment consensus mention that surgical operation is the first choice for the treatment of glioma.Because the purpose of the operation is to reduce the mortality,protect the function and improve the quality of life,the basic principle of surgical treatment is to remove the tumor on the basis of the function protection.The study of evidence based medicine shows that the degree of surgical resection is closely related to the prognosis of patients.[7]Because the glioma is located in the brain parenchyma,the tumor grows infiltrating and lack of visible and clear demarcation region,in clinical it is difficult to complete the full biological cut in the premise of protecting the brain function.The development of the operation mode of gliomas has gradually experienced total tumor resection under the naked eye,total tumor resection under the microscope and imaging total tumor resection.At present the development of surgery assistive technology including:(1)the application of positioning in preoperative functional imaging;(2)the application of neuronavigation in the surgery;(3)the application of intraoperative wake-up technology in operation;(4)ultrasound and MRI imaging technology;(5)the application of electric physiological detection in operation;(6)the application of fluorescent microscope and fluorescein angiography in operation.Alone or in combination with the application of above techniques,improve the accuracy of preoperative localization,realize to avoid the important functional areas in the operation,correct intraoperative brain shift,show residual tumor tissue,improve surgical safety and expand resection.However,the characteristics of the infiltration and diffuse growth of gliomas decide that the simple operation could not complete all cut in Biology,postoperative tumor residual and recurrence is inevitable.Because of this limitation,on the basis of surgical treatment,the adjuvant therapy such as radiotherapy,chemotherapy,immunotherapy,targeted therapy,gene therapy has been developed.Radiotherapy is an important adjuvant therapy for the treatment of gliomas,and its therapeutic effect has long been recognized and affirmed,and mamy studies have indicated that postoperative radiotherapy can improve the prognosis.[8,9,10]At first the radiotherapy for primary gliomas is the whole brain radiation therapy,and the side effects are brain radiation injury.In recent years,with the advancement of radiation technology,the whole brain radiotherapy gradually shifts to local radiotherapy.The aim is to reduce the short-term and long-term normal brain tissue damage,improve the prognosis,and improve the quality of life while as far as possible to inhibit tumor cells.At present,there are Stereotactic radiotherapy,3D-conformal radiation therapy(3D-CRT),Intensity modulated radiation therapy(IMRT)and so on.Chan and other’s research shows that the new radiotherapy technology does not improve the treatment effect of tumor.[11,12]At present,the main function of precise radiotherapy is to reduce the radiation damage of normal brain tissue.Because of the difference of the sensitivity of glioma cells to radiotherapy,the effectiveness of radiotherapy is not guaranteed.The improvement of the efficacy of early conventional chemotherapy drugs in the treatment of glioma is very limited.In 2005,the new England Journal of Medicine published the results of the study of the concurrent radiotherapy and chemotherapy of temozolomide(TMZ)in the treatment of gliomas,which has an epoch making significance in the history of chemotherapy and radiotherapy for gliomas.[6]On the basis of this,the Stupp scheme has become the standard treatment for the treatment of glioblastoma.Although the emergence of TMZ has made a great progress in the treatment of glioma,but its inherent toxic side effects,as well as not through the blood brain barrier,drug resistance and other defects,limiting its application and efficacy.Currently,targeted therapy is mainly targeted at cell proliferation,cell surface receptors related to angiogenesis and signal transduction factors.Mainly include:growth factor and receptor inhibitors,cell signal transduction inhibitors,anti angiogenic drugs,etc..But at present,there are no significant effects on the prognosis of gliomas,and the combined application of various programs is needed.The incidence rate of glioma is high,the degree of malignancy is high,the prognosis is poor,the treatment effect is not satisfied.Human malignant glioma is a serious challenge for all treatments.Therefore,the research and development of new promising antiglioma drugs become urgent needs.[13]Flavonoids are the most important and largest class of plant phytochemicals present in a variety of fruits,vegetables and beverages of plant origin.Flavonoids have been reported to exhibit antioxidant,anticancer and antimutagenic activities.[14]These phytochemicals are believed to have a significant protective role in several chronic diseases including malignant tumors.This also explains the well-known relations between decreased risk of numerous cancers and high consumption of fruits and vegetables containing flavonoids.[15,16,17,18,19]Chalcone structure comprises of an open-chain flavonoid with two aromatic rings connected by a 3-carbon fragment bearing a,3-unsaturated carbonyl motif.They are isolated from the plants and are believed to be the synthons of natural products like flavonoids and isoflavonoids.[20]Tomato is a popular plant,is planted almost all over the world.Tomato contains a variety of pophenols,such as chlorogenic acid and rutin.Naringenin chalcone also belong to the flavonoid phytochemicals found in red tomato peel.[21]To the best of our knowledge,there are no such reports on the antiglioma activity of naringenin chalcone reported in the published literature.So the aim of the present investigation was to study the antitumor potential of naringenin chalcone in U87MG human glioblastoma cells and in xenograft mice model along with evaluating its effects on apoptosis,autophagy and PI3K/Akt pathway.ObjectiveTo investigate the in vitro and in vivo anti-glioma effects of naringenin chalcone in U87MG glioma cells and xenograft model.MethodsThe first test is to investigate the vitro antitumor effects of naringenin chalcone on U87MG glioma cells.U87MG glioma cells with different concentrations(0,2.5,5,10,50,75 and 100μM)naringenin chalcone,different culture time(24,48,72h),respectively by MTT cell proliferation assay.U87MG glioma cells with different concentrations(0,10,50 and 100μM)naringenin chalcone after cultured for 48 hours,respectively by acridine orange/ethidium bromide and Hoechst33342 staining under a fluorescence microscope to assess cell apoptosis.U87MG glioma cells with different concentrations(0,10,50 and 100μM)naringenin chalcone after cultured for 48 hours,observe the number and size of autophagic vacuoles in cells under transmission electron microscope.U87MG glioma cells with different concentrations(0,10,75 and 100μpM)naringenin chalcone after cultured for 48 hours,Annexin V-FITC and PI stain after treatment,use flow cytometry to detect U87MG human glioma cell early apoptosis and late apoptosis.U87MG glioma cells are starved for 4 hours in serumfree medium,and then add different concentrations(0,10,50 and 100μM)naringenin chalcone and culture for 40 minutes and 48 hours,use the Western blot to evaluate the effect of naringenin chalcone on PI3K/Akt signal transduction protein.Use a mouse xenograft model to evaluate the in vivo anti-tumor effect of naringenin chalcone on U87MG glioma cells.The U87MG human glioblastoma cells are subcutaneously injected into the right rear flank of each mouse to produce tumors in mice.After tumor development,the mice are divided into 5 groups and are given different doses of naringenin chalcone injection(5,20,40 and 80 mg/kg).24 days later,the mice are killed,the weight,length and width of the tumor are measured,and the tumor volume is calculated.ResultsNaringenin chalcone has inhibitory effect on proliferation of U87MG cells,the cytotoxicity increase with concentration and time.That is to say,the cytotoxic effect of naringenin chalcone on U87MG cells is with dose and time dependent.The naringenin chalcone treated U87MG cells are stained by acridine orange/ethidium bromide under fluorescence microscopy,are observed the formation of cell shrinkage and apoptosis.After Hoechst 33342 staining,chromatin condensation,DNA fragmentation,cytoplasmic vacuoles are observed under the fluorescence microscope.These are the morphological characteristics of apoptosis.Under transmission electron microscopy it shows that the naringenin chalcone can induce the formation of autophagic vacuoles,and the number and volume of autophagic vacuoles increases with naringenin chalcone dose increases.Detected by flow cytometry,naringenin chalcone can induce U87MG glioma cell early apoptosis and late apoptosis.Naringenin chalcone can also inhibite phosphorylated and non phosphorylated PI3K and Akt protein,inhibite NF-κB、Bcl-2,activite Caspase-3.In vivo test,the results show that the tumor volume and weight treated with different doses of naringenin chalcone are less than the control group.Conclusionnaringenin chalcone exhibits antitumor activity in U87MG human glioblastoma cells and in xenograft mice model by inducing apoptosis,autophagy and PI3K/Akt pathway.更多还原