【作者】 付元山；

【导师】 李宏；

【作者基本信息】 大连医科大学 ， 病理学和病理生理学， 2010， 博士

【摘要】 背景与目的：髓母细胞瘤为好发于儿童期的中枢神经系统恶性肿瘤,具有快速增殖和高侵袭性的特点,是小儿死亡率最高的恶性肿瘤。该肿瘤大多出现在后颅窝的小脑蚓部,起源于小脑神经元前体细胞。常规治疗方案是在发现后进行手术切除辅以术后的化疗和／或放疗。近年来随着肿瘤准确定位水平的提高以及手术方式和放化疗方案的优化,改善了治疗的效果,患者生存率有一定程度的延长。然而,术后的高复发率和放化疗给患儿带来包括神经精神障碍等严重毒副作用,这己成为神经肿瘤学的治疗难题并明显降低了患者的生存质量。髓母细胞瘤属于原始神经外胚层肿瘤(primitive neuroectodermal tumors, PNET),是由于小脑神经干细胞增殖异常和缺乏终末分化而导致。在正常小脑发育过程中诸多信号通路都参与调控,在受到外源性信号影响后部分信号通路的异常使得前体细胞持续增殖且成熟阻碍,从而导致髓母细胞瘤的发生。由此可见,在合适的分化诱导剂的作用下,髓母细胞瘤有可能获得继续分化的潜能。因此,安全可靠的分化诱导剂可为髓母细胞瘤的化学治疗提供新的途径。研究表明,某些药物能够诱导髓母细胞瘤细胞分化并使其出现神经元或神经胶质细胞的形态学特点和相应的生物标志物。我们的前期研究结果显示,广泛存在于葡萄等天然食物中的白藜芦醇能够抑制髓母细胞瘤细胞生长,以剂量相关性方式诱导肿瘤细胞分化和凋亡：同时,伴有神经分化早期标志物神经配基1(Neurogeninl, NGN1)和突触素(Synaptophysin, SYP)的表达和产生以及与神经发育和肿瘤形成有关的信号转导通路的活性改变。有关该化合物在中枢神经系统的脑肿瘤中的代谢形式和生物利用度正在探讨中。与白藜芦醇不同,全反式维甲酸是临床常规使用的分化促进剂。它是维生素A的衍生物,能够抑制髓母细胞瘤细胞的生长,促进其分化,在胚胎发育、器官形成、精子产生、视觉和细胞分化中发挥广泛生物学效应,人体在动物食品如肉类和植物食品如胡萝卜中摄取获得。早期研究发现,维生素A缺乏的动物具有肿瘤的易感性。实验和流行病学研究也从多方面证实维甲酸活性缺失和反应性降低与多种肿瘤的发生具有相关性。同时,天然的或人工合成的维甲酸类药物在肿瘤动物模型中有高效抑制肿瘤发生的作用。肿瘤的发生包括细胞转化为非正常的强增殖能力的、去分化的细胞,由于维甲酸能够抑制细胞增殖,促进分化而在肿瘤化学治疗中作为一线促分化剂使用,并且在部分肿瘤中尤其是急性早幼粒性白血病中获得良好的效果。本实验室前期的研究结果也验证和支持前述的结论,维甲酸的促分化作用与凋亡基因Fas和白血病抑制因子(leukemia inhibitory factor,LIF)的表达,端粒酶的活性均相关。但是,本实验室以及其它学者的研究也表明,维甲酸对髓母细胞瘤的作用有明显的差异,即部分髓母细胞瘤出现增殖抑制和分化倾向而其它则对维甲酸的反应性差甚至没有反应。迄今为止,尚未阐明导致这种现象的内在分子机制。业已发现,维生素A(视黄醇)进入人体后转变成视黄醛,再经氧化变成全反式维甲酸(All trans retinoic acid, RA)。维甲酸的药理作用通过其信号传导通路实现：维甲酸脂溶性特点使得外源性维甲酸直接由细胞外基质透过细胞膜进入胞浆,而内源性维甲酸则由视黄醛脱氢酶2(retinaldehyde dehydrogenase type 2, RALDH-2)在细胞浆内合成。细胞内的维甲酸有两种命运：被代谢酶CYP26A1 (cytochrome P450 26A1, CYP26A1)分解为极性产物而排出胞外,或与胞浆内的维甲酸受体结合蛋白2(cellular retinoic acid binding protein 2, CRABP-Ⅱ)结合进而入核与维甲酸受体(retinoid acid receptor and retinoid x receptor, RAR andRXR)形成复合物。维甲酸受体是核内转录因子,能与下游基因的启动子区域的维甲酸反应元件(retinoic acid response element, RARE)结合,引发相应基因的转录,最终实现维甲酸的生物学效应。可见,CRABP(包括两种异构体CRABP-Ⅰ和-Ⅱ)、CYP26A1和维甲酸受体各亚型在维甲酸信号转导中起主要的作用；其中,CRABP-Ⅰ的功能主要是提高CYP26A1对维甲酸的代谢效率。维甲酸受体RAR和RXR都有3种亚型,即RARα/RARβ/RARγ和RXRα/RXRβ/RXRγ,它们以二聚体的形式出现并发挥作用。诸多研究结果表明,肿瘤形成的过程伴随着多个信号转导途径的异常与紊乱。与Notch, Wnt等信号通路相似,维甲酸信号通路不但在神经发育中发挥作用,还与髓母细胞瘤和其他肿瘤如白血病等的发生密切相关,该通路广泛参与细胞命运选择,影响细胞的增殖和分化。维甲酸受体与其配体结合而致的维甲酸信号通路活化能够促进肿瘤细胞的分化和凋亡,但该作用具有细胞类型选择性。诸多肿瘤细胞对维甲酸的反应差异还表现在有些细胞在药物作用下可以分化甚至凋亡,而在另一些肿瘤细胞则无类似现象。本实验室前期的研究表明,髓母细胞瘤细胞系Med-3在维甲酸处理后细胞出现分化,而UW228-2细胞系则形态和生长方式依旧。利用不同维甲酸敏感性的髓母细胞瘤细胞系进行深入研究,有助于揭示肿瘤细胞维甲酸敏感和耐受的内在机制,对临床个性化用药和改善患者预后具有重要的意义已知,维甲酸信号通路的状态与肿瘤的维甲酸敏感性有关。经典的维甲酸信号通路组分异常如在急性早幼粒性白血病中RARα基因与早幼粒性白血病基因(promyelocytic leukemia, PML)发生了融合,会引起早幼粒细胞正常的成熟分化受阻、抑制肿瘤抑制基因表达和PML的促凋亡功能,促使急性早幼粒性白血病(Acute promyelocytic leukemia, APL)的发生,而RA能够靶向结合到癌蛋白PML-RARα中的维甲酸受体结构域,重新启动髓系细胞的分化基因调控网络,诱导白血病细胞分化继而凋亡；Hu和Gebert等学者对口腔上皮鳞状细胞癌和肺癌研究发现,癌变细胞中RARP2基因表达异常并对维甲酸治疗的敏感性消失。RARγ选择性结合维甲酸更大程度上以诱导凋亡,而不是促进分化的形式抑制神经母细胞瘤生长；甲状腺正常组织中RXRγ表达缺失,甲状腺癌组织中出现RXRγ的过表达。而用9-cis RA处理RXRγ过表达的甲状腺癌细胞系和不表达RXRγ的甲状腺癌细胞系细胞后,前者出现生长抑制和凋亡,后者则无此作用。CRABP-Ⅰ可使RA的降解增加,导致进入核内与核受体结合的RA数量减少,这样就使头颈部鳞癌细胞对RA产生耐药；在人的乳腺上皮细胞中,AP2因子可通过调节CRABP-Ⅱ基因的表达水平来调控细胞对RA的反应能力。包括CYP26A1在内的其他维甲酸信号组分表达改变同样影响着维甲酸的效应。以上说明,维甲酸信号通路的异常在肿瘤中以不同形式单一或组合出现,有必要从维甲酸信号通路的整体性角度对髓母细胞瘤的维甲酸敏感性差异进行系统的研究。然而,迄今为止国内外尚无有关维甲酸通路在髓母细胞瘤中的状态及其与化疗敏感性的报道。为此,本研究以髓母细胞瘤细胞系和肿瘤组织为研究对象,分析维甲酸信号通路在不同维甲酸敏感性的髓母细胞瘤细胞系中的状态以及维甲酸信号通路组分的表达差异与维甲酸敏感和耐药的关系；在此基础上,进一步探讨导致维甲酸信号通路组分基因沉默的表观遗传学机制,以期为改善髓母细胞瘤临床治疗策略提供科学依据。材料与方法：髓母细胞瘤及瘤旁相对正常小脑组织来由大连医科大学第一附属医院病理科和中国医科大学盛京医院病理科提供。实验所用人髓母细胞瘤细胞系Med-3由日本神户大学神经外科建立和提供,另外三个细胞系UW228-1、-2和-3由华盛顿大学(西雅图)神经肿瘤学实验室提供。本研究采用石蜡组织微阵列,细胞培养、免疫组织化学(IHC)、免疫细胞化学(ICC)、反转录聚合酶链式反应(RT-PCT)、蛋白印迹(Western-blotting)、甲基化PCR(MP)、DNA测序和RNA干扰(RNAi)等方法,开展了以下实验内容：1)观察维甲酸信号通路组分RARα/β/γ、RXRα／β／γ、CRABP-Ⅰ／-Ⅱ和CYP26A1等在维甲酸敏感(Med-3)与耐药(UW228-2)的髓母细胞瘤细胞系中的表达情况以及维甲酸对它们转录水平的影响；2)检测CRABP-Ⅰ和-Ⅱ基因表达异常与启动子区域甲基化的关系；3)使用去甲基化剂(5-Aza)使沉默的CRABP-Ⅱ恢复表达,而后观察观察维甲酸后对被处理细胞的生长、分化和凋亡的影响：4)使用CRABP-Ⅱ特异性siRNA转染维甲酸敏感的Med-3细胞系并观察其敏感性的变化；5)通过髓母细胞瘤石蜡组织微阵列结合免疫组化技术检测CRABP-Ⅰ／-Ⅱ和突触素(Synaptophysin)的表达情况；实验数据采用SPSS12.0软件包应用斯皮尔曼等级相关(Spearman Rank Correlation)和双因素相关性(Bivariate Correlation)检验方法进行统计分析结果：一.维甲酸信号通路组分在敏感与耐药的髓母细胞瘤细胞系中的表达状态以及维甲酸对它们转录水平的影响1.HE染色结果、流式细胞分析和部分RT-PCR结果：验证了维甲酸对不同髓母细胞瘤细胞系作用的差异,敏感的Med-3细胞系在维甲酸处理后形态出现神经元样分化改变以及神经元分化早期标志物NGN1出现表达,药物作用后出现S期阻滞。而细胞系UW228-2则未观察到上述现象。2.MTT分析和死活细胞比分析结果：维甲酸处理Med-3细胞系48h和72h,与正常培养细胞OD值比较,前者为p=0.006,后者p=0.001,统计分析有显著意义,UW228-2细胞系在24h,48h和72h与正常培养细胞OD值比较均无统计学意义；维甲酸处理48h和72h的Med-3细胞系的死活细胞比与正常培养细胞间存在显著性差异(p=0.000),有显著统计学意义。UW228-2细胞在维甲酸作用24h,48h和72h的死活细胞数与正常培养细胞相比无统计学意义。3.RT-PCR结果显示：髓母细胞瘤UW228-2细胞存在CRABP-Ⅰ和-Ⅱ的表达缺失且与药物处理无关；而维甲酸敏感的Med-3细胞在正常培养条件下有低水平表达,药物作用后其表达水平明显提高。4.蛋白印迹和免疫细胞化学染色结果在蛋白水平进一步证实UW228-2细胞的CRABP-Ⅰ／-Ⅱ表达缺失和维甲酸对Med-3细胞两基因表达的可调节作用。二.启动子区高甲基化导致CRABP-Ⅱ基因沉默和UW228-2细胞系维甲酸耐药的内在分子机制1.亚硫酸盐修饰后的甲基化PCR结果显示：CRABP-Ⅱ出现电泳条带而CRABP-Ⅰ则无；对扩增产物所做的DNA测序发现,CRABP-Ⅱ基因启动子区域有14个CpG岛位点出现甲基化。2.去甲基化处理后的UW228-2细胞中CRABP-Ⅱ恢复表达且其表达水平与去甲基化剂5-Aza的使用剂量呈正相关；相反,在同样处理条件下CRABP-Ⅰ仍未恢复转录；另外,在维甲酸处理前后低水平表达的RARα和RARβ也未见转录增强。3.去甲基化处理后的UW228-2细胞对RA的敏感性增强并出现向神经元分化的倾向,伴有与神经分化早期标志物NGN1和β-Ⅲtubulin的表达或表达上调和细胞凋亡。死活细胞比分析的结果表明,在24h,36h和48h三个时间点,去甲基化后单用RA组和去甲基化联合RA组与正常培养组、正常培养加RA组和单纯去甲基化组的细胞数比较,p值均小于0.05。干扰可降低Med-3细胞的维甲酸敏感性CRABP-ⅡRNAi三.1.针对CRABP-ⅡmRNA不同位点设计的siRNA获得了较好的抑制效率,最高达83%；转染试剂的转染效率可达90%以上。2.CRABP-Ⅱ-RNAi使Med-3细胞出现维甲酸耐受2.1正常培养组,错配序列组,干扰组和干扰后加维甲酸组分别在24h,48h,72h,96h,120h5个时间490nm波长点,在酶联免疫监测仪上测定各孔光吸收值。结果显示,四组间在各时间点比较无统计学差异(p>0.05)。2.2 CRABP-Ⅱ-RNAi持续48小时后续用维甲酸72小时的HE染色显示,Med-3细胞未出现分化表型和凋亡征象,而免疫细胞化学显示CRABP-Ⅱ蛋白在干扰后出现明显下降。2.3对上述被处理细胞所做的流式细胞术检测表明无明显细胞周期阻滞且正常培养和CRABP-ⅡRNAi转染组细胞凋亡指数相近。四.髓母细胞瘤和瘤旁组织中CRABP-Ⅰ／-Ⅱ和synaptophys in的表达104例髓母细胞瘤组织中小细胞型有58例,大细胞型(包括间变型)40例,结节型(包括促结缔组织增生型)6例。所有瘤旁组织中CRABP-Ⅰ／-Ⅱ和synaptophysin表达均呈阳性表达,肿瘤组织中的表达情况如下：1.CRABP-Ⅱ在髓母细胞瘤组织中的表达CRABP-Ⅱ阳性染色为细胞核中呈现粗细不一的棕黄至棕褐色颗粒。在104例髓母细胞瘤组织中,有45例呈阳性表达(+-++,43.3%),混合阳性(+／-)有15例(14.4%),其余为阴性44例(42.3%)。在45例阳性表达的组织中,小细胞型有24例,占该型的41.4%；大细胞型19例,占该型的47.5%；结节型2例,占该型的33.3%。2.CRABP-Ⅰ在髓母细胞瘤组织中的表达CRABP-Ⅱ以细胞浆中出现棕黄色至棕褐色颗粒为阳性染色。在104例髓母细胞瘤组织中,有39例呈阳性表达(+-++,37.5%),混合阳性(+／-)有5例(4.8%),其余为阴性。在39例阳性表达的组织中,小细胞型有20例,占该型的34.5%；大细胞型19例,占该型的47.5%；结节型未见阳性表达。3.Synaptophysin在髓母细胞瘤组织中的表达Synaptophysin阳性染色为细胞浆中呈现棕色至棕黄色颗粒。在104例髓母细胞瘤组织中,有74例呈阳性表达(+-++,71.1%),混合阳性(+／-)有6例(5.8%),其余为阴性24例(23.1%)。在74例阳性表达的组织中,小细胞型有38例,占该型的65.5%；大细胞型33例,占该型的82.5%；结节型3例,占该型的50%。4.CRABP-Ⅱ与synaptophysin表达的关联度分析应用斯皮尔曼等级相关(Spearman Rank Correlation)检验方法进行统计分析,结果表明：104例髓母细胞瘤组织CRABP-Ⅱ和synaptophysin的表达呈正相关(rs=0.317,p=0.001),即synaptophysin表达阳性的组织,CRABP-Ⅱ有更大的可能性也是呈阳性表达,反之亦然。肿瘤各亚型中两种蛋白表达关联分析说明在小细胞型中CRABP-Ⅱ和synaptophysin的表达呈正相关(rs=0.576,p=0.000),而在大细胞型中这种关联并不存在(rs=0.09,p=0.579)。CRABP-Ⅰ和-Ⅱ的叠加分析说明二者均表达沉默的在小细胞型中有18例,占31.0%；在大细胞型中有10例,占25.0%,结节型中占3例,占50%。CRABP-Ⅰ和-Ⅱ表达的双因素分析表明两者间无相关性(p>0.05)结论：1.髓母细胞瘤细胞对维甲酸存在敏感性差异,Med-3为对维甲酸敏感而UW228-2为对其耐药的细胞系。2.髓母细胞瘤的维甲酸敏感性差异与其信号通路组分尤其是CRABP-Ⅱ表达的差异有关。3.CRABP-Ⅱ基因启动子区域CpG岛高甲基化是导致其沉默的内在机制。4.通过去甲基化恢复CRABP-Ⅱ基因表达能提高UW228-2细胞对维甲酸的敏感性。5.CRABP-Ⅱ-RNAi能够高效抑制CRABP-Ⅱ的表达并使维甲酸敏感的髓母细胞瘤细胞系Med-3对维甲酸的反应性降低。6.CRABP-Ⅱ表达调节实验说明,该基因的表达与否与是决定维甲酸抗癌效果的重要因素。7.首次揭示,髓母细胞瘤组织中的CRABP表达存在阳性,阴性和阴阳混合3种形式,该基因多样性和异质性表达可能是造成维甲酸对髓母细胞瘤疗效显著性差异以及维甲酸首发和继发耐药的重要原因。以上研究结果为维甲酸在髓母细胞瘤治疗中的合理和个性化用药提供有价值的实验依据,具有明显的转化肿瘤学意义更多还原

【Abstract】 Backgrounds and Objectives:Medulloblastoma (MB) is the most common malignant tumour of central nervous system (CNS) during childhood with high invasiveness and mortality. It is originated from precursors of cerebellar neurons and occurs in most cases in vermiform of cerebellum which is located in posterior cranial fossa. The conventional therapy was surgical removal combined with chemo-and/or radiotherapy. For the past few years along with improvement of tumor pinpoint and optimization of surgical types and chemoradiation scheme, curative effect and survival rate were somewhat ameliorated. But the high recurrence rate, short-and long-term adverse effects includes neurological and/or mental disorders of conventional postsurgical adjuvant therapies have become therapeutic problem of neurooncology, which also lower the patient’s living quality.MB belongs to primitive neuroectodermal tumors (PNET) and results from abnormal cell proliferation and deficient terminal differentiation. Normal cerebellum development is regulated by many signaling pathways. The development of MB is related with abnormalities of some signaling pathways which promote continuous proliferation of precursors and mature obstruction. So that, MB seems to keep the potential to return to differentiation route if being treated by suitable inducer(s), which may be provide new chemotherapy method. It has been proved that some drugs induce differentiation of MB cells in terms of neuron-like phenotype and expression of neuron biomarkers. Our previous studies indicated that resveratrol, a polyphenolic compound existed in grapes and many other natural foods, exhibited ideal anti-medulloblastoma effects by promoting either differentiation or apoptosis in time-and dose-dependent fashions, followed by the appearance of neuronal biomarkers such as neurogeninl (NGN1) and synaptophysin (SYP) and the activation of multiple signaling pathways related with neural development and tumorgenesis. The metabolic style and bioavailability of resveratrol in brain tumors of CNS is in investigation in order to identify internal molecular mechanism.In difference with resveratrol, all-trans retinoic acid (RA) is conventionally used differentiation-promoting agent in clinic. RA, the derivative of vitamin A, could inhibit growth of MB cells and facilitate differentiation. It has widespread biological effect in embroyo development, spermgenesis and visual development which obtained from animal food such as meat and vegetable food such as carrot. Vitamin A deficiency animals were susceptible to cancers in earlier studies. Meanwhile natural or artificial retinoids could inhibit tumorigenesis efficiently. Tumorigenesis accompanys with transformation of normal cells into abnormal strong proliferating and dedifferentiating ones. Because of the growth inhibitory and differentiation promoting effects, RA was used as first line differentiation-promoting agent in cancer chemotherapy in the clinical management of acute promyelocytic leukemia (APL). Earlier studies in our lab also proved and supported the above mentioned conclusion. Moreover, the RA differentiation-promoting function was correlated with apoptotic related gene-Fas, leukemia inhibitory factor (LIF) and telomerase activity. Nevertheless, the studies of our lab and others also indicated that the thera-peutic effects of RA were distinct in different MB cells which respond with growth-inhibition and differentiation or weakly or no response. So far the internal molecular mechanism underlying that phenomenon has not been clarified.It has been already proved that vitamin A (retinol) should turns into retinaldehyde after body admission, then convert into RA by oxidation. RA’s pharmacologic function was implemented by its signaling pathway. Liposolubility makes extrinsic RA permeating from extracellular matrix into cytoplasm directly and exdogenous RA is synthesized by retinaldehyde dehydrogenase type 2(RALDH-2) in cytoplasm. Intracellular RA has two fates, one is decomposed by (cytochrome P450 26A1/CYP26A1) into polar products which will be eliminated out of cells, the other is to bind with cellular retinoic acid binding protein 2 (CRABP-Ⅱ) and form complex with retinoid acid receptor and retinoid x receptor (RAR and RXR) after transmitting from cytoplasm into nucleus. RAR and RXR, the intranuclear transcription factor, could induce transcription by binding with retinoic acid response element (RARE) within the promoter of the downstream genes. It is thus clear that CRABP, includes 2 isomers-Ⅰand-Ⅱ, CYP26A1 and RAR/ RXR subtypes play principal roles in transduction of RA signals. The main function of CRABP-Ⅰis to promote metabolic efficiency of CYP26A1. Both RAR and RXR have 3 subtypes, RARα/β/γand RXRα/β/γwhich function as dipolymer.A body of evidence shows that tumorigenesis is process of dysfunction and abnormality of one or more signaling pathways. Similar with the pathways mediated by Notch and Wnt, RA signaling pathway not only plays important roles in neural development but also links with the development of MB and other cancers such as leukemia. This signaling participates in cell fate decision through impact on proliferation and differentiation. Activation of RA signaling pathway induced by RAR/RXR binding with ligand promotes differentiation and apoptosis of cancers in cell type related fashion. Our previous work indicated that Med-3, a MB cell line, showed differentiation tendency after RA treatment. But UW228-2, another MB cell line, showed little response to RA treatment. The differential responses of MB cells to RA thus provide an ideal experimental model for exploring the underlying molecular mechanism leading to RA sensitivity or resistance. It has important significance for personalized RA therapy and prognosis-promoting in clinic.It has been known that the state of RA signaling pathway is correlated with its chemosensitivity. Classical abnormality of RA signaling compo-nents is RARa form fusion protein with promyelocytic leukemia (PML) which result in mature blockade of premyelocyte, inhibiting expre-ssion of tumor suppressor gene and PML’s apoptosis-promoting function. RA could restart differentiation-regulating network by binding to RAR domain of oncoprotein PML-RARa which induce leukemic cells different-tiation or even apoptosis. The studies performed on oral squamous cell carcinoma revealed that decreased RARβ2 in cancer cells associated with loss of RA sensitivity. The binding of RA with RARy selectively inhibits growth and induces differentiation and apoptosis of neuroblastoma cells. RXRy was deficient in normal tissue of thyroid gland and overexpression in some thyroid cancers. If treated with 9-cis RA, the growth of thyroid cancer cells with RXRy over-expression was inhibited and subjected to apoptosis. In contrast no change could be observed in the thyroid cancer cells that RXRy silenced. In human mammary epithelial cells, CRABP-Ⅱ, which could regulate RA reaction ability, was regulated by AP2 factor. Other compo-nents of RA signaling pathway includes CYP26A1 also influence the effects of this drug. In view of the above, different types of abnormality of RA signaling in tumors, single or combination, makes it necessary to study on different RA sensitivity in MB from entirety of this pathway. So far no report was seen about entire RA signaling pathway on MB cells and tissues. In this respect by studying on MB cells and tissue, we analyze the state of RA signaling pathway components in different sensitivity MB cells and the correlation with RA effects, sensitive or resistant. On this basis, epigenetic mechanism which cause gene silence of RA signaling pathway was investigated for the purpose of providing reasonable base for improvement of MB therapeutic strategy.Materials and Methods:MB and tumor-surrounding noncancerous cerebella were provided by Department of pathology, the 1st affiliated hospital of Dalian Medical University and Shen-Jing hospital of China Medical University. Med-3 MB cell line was kindly provided by the doctors in the Department of neurosurgery, Kobe University School of Medicine and UW228-2 cell line was established and provided by the Department of neurological surgery, University of Washington at Seattle. By the methods of paraffin embedded tissue array immunohistochemistry(IHC), cell culture, immunocytochemistry(ICC), reverse transcription-polymerase chain reac-tion(RT-PCR), Western blotting(WB), mythylation-polymerase chain reaction(MP), DNA sequencing and RNA interference(RNAi), the following experiments were performed:1) The expression of RARaα/β/γ、RXRaα/β/γ、 CRABP-Ⅰ/-Ⅱand CYP26A1 was detected in RA sensitive Med-3 cell line and RA resistant UW228-2 cell line. Transcription level change of them after treatment of RA were also studied.2) The correlation between promoter methylation and abnormal expression of CRABP-Ⅰand-Ⅱwas explored.3) Demethylation agent(5-Aza) was used to recover expression of silenced CRABP-Ⅱand then RA effects on growth, differentiation and apoptosis was observed.4) Directed siRNA of CRABP-Ⅱwere applied to transfect RA sensitive Med-3 cells and change of RA sensitivity were determined.5) By combination of paraffin embedded tissue array and IHC, CRABP-Ⅰ/-Ⅱand synaptophysin expression were analyzed. The data were statistically analyzed by Spearman Rank and Bivariate Correlation with SPSS 11.0 software.Results:1. The status of RARα/β/γ、RXRα/β/γ、CRABP-Ⅰ/-Ⅱand CYP26A1 in RA sensitive and resistant MB cell lines and transcription level change of them after treatment of RA.1.1 By HE staining and Flow cytometry analysis, different RA chemosen-sitivity in variant MB cell lines were studied. Neuron-shaped change and appearance of NGN 1, primary differentiating marker, were observed in RA sensitive Med-3 cell. RA treated Med-3 cell exhibited s-Phase arrest.1.2 Statistic analysis of OD value between normal and RA treated Med-3 cell for 48h and 72h is significant(p=0.006 and p=0.000).Oppositely Statistic analysis of OD value between normal and RA treated UW228-2 cell for 24h,48h and 72h,, there was no statistical difference. The difference of viable/nonviable ratio between normal and RA treatd Med-3 cell at time point 48h and 72h was significantly(p=0.000), but that in UW228-2 was insignificantly(p>0.05).1.3 By RT-PCR expression of CRABP-Ⅰ/-Ⅱwas lost in UW228-2, no matter RA treatment or no. Low level transcription of these two genes was observed in RA sensitive Med-3 cell, markedly elevated after RA addition.1.4 Further WB and ICC confirmed that lost of CRABP-Ⅰ/-Ⅱin UW228-2 and expression of them in Med-3 is regulable.2. Promoter hypermethylation induce silence of CRABP-Ⅱand RA resist-ance in UW228-2.2.1 Methylation PCR (MP) results showed that CRABP-Ⅱbut no CRABP-Ⅰ band could be observed at corresponding molecular weight location. Bisulfite sequencing indicated that there were 14 sites methylation of CpG islands.2.2 CRABP-Ⅱbut not CRABP-Ⅰexpression was recovered by 5-Aza, a demythylation agent, in dose-related fashion. Meanwhile no elevation was observed in RARa and RARβtranscription level after demethylation.2.3 RA sensitivity was overturned in resistant UW228-2 after 5-Aza treatment. Cell growth was inhibited. Apparent neuron-like morphology and cell death was induced. Furthermore, NGN1 andβ-Ⅲtubulin, the early neuronal differentiation biomarker, was expressed or in higher. Statistical analysis of MTT assay indicated that the difference between demythelation following with RA treatment groups(one group is single RA treatment after demethylation, the other group is that combined RA and 5-Aza treatment after demethylation.) and control groups was significantly (p<0.05).3. CRABP-ⅡsiRNA reduced RA-sensitivity of Med-3 cells1. Three designed siRNA aimed directly at different sites silenced CRABP-Ⅱat.different level, the highest inhibition ratio was 83%. The transfection efficiency of transfection agent was above 90%.2. CRABP-Ⅱ-RNAi makes the RA sensitive Med-3 cell resistant.2.1 No statistical significance of OD values from MTT assay among the four experimental groups (p>0.05) includes normal culture condition (Normal), transfected with Mock siRNA (Mock) or CRABP-Ⅱspecific siRNA (siRNA) and treated by CRABP-Ⅱspecific siRNA for 48 hours followed by 10μM RA treatment starting at 48 hour time point (siRNA+RA).2.2 CRABP-Ⅱoriented immunocytochemical staining results indicatd that it was markedly decrease after RNAi. H & E staining performed on normally cultured Med-3 cells and the cells treated by siRNA and RA combination showed that no differentiation and apoptosis were observed.2.3 Flow cytometry analysis of Med-3 cells harboring CRABP-ⅡsiRNA without and with 3 day 10μM RA treatment proved that no obvious cell cycle arrest and apoptosis index change.4. Expression of CRABP-Ⅰ/-Ⅱand Synaptophysinin in MB and tumor-surrounding noncancerous cerebella.In 104 MB tumor tissues,58 samples were diagnosed as the classic subtype,40 as large cell subtype(includes anaplastic ones) and 6 as nodular subtype(includes desmoplastic ones). CRABP-Ⅰ/-Ⅱand Synaptophysinin were constitutively expressed in 12 tumor-surrounding noncancerous cere-bella. Expression of them in MB tissues is as follows:4.1 Positive staining of CRABP-Ⅱwas brown in nucleus.45 (45/104,43.3%) of the 104 tumor samples were positive,44 (44/104,42.3%) negative and 15 (15/104,14.4%) partly positive in CRABP-Ⅱexpression.58 samples were diagnosed as the classic subtype,40 as anaplastic/large cell subtype and 6 as nodular subtype, and the above CRABP-Ⅱstaining patterns could be observed among the classic, anaplastic/large cell and nodular subtypes. In 45 CRABP-Ⅱpositive samples,24(24/58,41.4%) of them were classic, 19(19/40,47.5%) were large cell subtype and 2(2/6,33.3%) were nodular ones.4.2 Positive expression of CRABP-Ⅰwas brown particles in cytoplasm. 39(39/104,37.5%) of the 104 MB tissues were positive,5 (5/104,4.8%) partly positive and others (60/104,57.7%) negative in CRABP-Ⅰexpression. In 39 CRABP-Ⅰpositive samples,20(20/58,34.5%) of them were classic, 19(19/40,47.5%) were large cell subtype and no CRABP-Ⅰpositive staining was observed in nodular subtype.4.3 To synaptophysin, brown particles in cytoplasm represents positive staining.74(74/104,71.1%) of the 104 MB samples were positive, 24(24/104,23.1%) negative and 6 (6/104,5.8%) partly positive in synaptophysin expression. In 74 synaptophysin positive samples,38(38/58, 65.5%) of them were classic,33(33/40,82.5%) were large cell subtype and 3(3/6,50%) were nodular subtype.4.4 CRABP-Ⅱand synaptophysin expression was closely correlated (Spear-man rank-correlation, rs=0.317; p=0.001) among the 104 medullobla-stoma tissues, and this correlation was more remarkable in the classic subtype (rs= 0.576; p=0.000). The overlapped CRABP-Ⅰand CRABP-Ⅱsilencing was found in 18/58 (31.0%) of the classic,10/40 (25%) anaplastic/large cell and 3/6 (50%) anaplastic/desmoplastic tumors and, therefore, showed no close correlation of their expression (p>0.05).Conclusion:1. RA chemosensitivity was different in MB cells, Med-3 was sensitive and UW228-2 resistant. 2. Different RA chemosentivity of MB was correlated with RA signaling pathway components especially CRABP-Ⅱexpression or no.3. CpG islands hypermethylation in promoter region of CRABP-Ⅱwas resp-onsible for its silence in MB.4. Expression recovery by demethylation of CRABP-Ⅱcould reverse RA resistant UW228-2 cell to sensitive.5. siRNA of CRABP-Ⅱcould silence its expression and makes the RA sensitive Med-3 cell resistant.6. Regulation experiments indicated that CRABP-Ⅱexpression or no determined RA anti-cancer effects.7. This study reveals for the first time that there were 3 types of CRABP expression, positive, partly positive and negative. The diversiform and heterogeneous CRABP expression was possiblely the key factor to explain markedly difference of RA effect on MB and important reason for primary and secondary resistance.8. These results will provide valuable experimental evidence for reasonable and personalized RA therapy in MB clinic and have apparent significance in translational cancer biology.更多还原