【作者】 王启宇；

【导师】 张红卫；

【作者基本信息】 山东大学 ， 发育生物学， 2008， 博士

【摘要】 当代生物学和医学研究的重要目标之一是增强对肿瘤的治疗效果,最终实现彻底治疗。化疗作为肿瘤治疗的主要手段,其作用机理决定了在发生作用时缺乏选择性,对患者产生全身性的毒副作用,限制了疗效的提高。因而改进治疗方法和开发副作用小的化疗新药等成为科研人员的重要课题。目前有关化疗的研究热点主要有:一、研究和开发特异的小分子化合物或生物活性分子,来特异性激活或关闭癌细胞中与增殖、凋亡和代谢有关的细胞信号通路活性,从而提高化疗药物的特异性,增强疗效。这是提高肿瘤化疗效果的最具希望和最有潜力的途径。二、通过传统化疗药物和新药(包括新开发的具有抑瘤效果的化学药物和天然药物)的相互作用,对患者联合用药,从而增强肿瘤细胞对化疗药物的敏感性(主要是克服肿瘤细胞对化疗药物的耐药性),这是提高肿瘤化疗效果的重要途径。三、通过改善用药方式,使药物通过各种手段直接作用于局部的实体瘤,并且缓慢释放,达到长效抑制肿瘤进而杀死肿瘤细胞的效果。研究显示,动物胚胎发育过程中的一些信号通路和肿瘤的发生和发展密切相关,如Wnt、Hh、Notch和BMP信号通路等。其中Hh信号通路和肿瘤的关系是近些年的研究热点之一。Hh信号通路不但在细胞命运的决定和组织图式的形成中起到至关重要的作用,而且在肿瘤发生和发展中也起到关键作用。近年来,Hh信号通路在人类肝细胞癌(HCC,Hepatocellular caner)中的作用和意义正逐步为研究者所重视。有研究表明,Sonic Hedgehog(SHh)信号通路在HCC中是高频率激活的,并参与了HCC的发生和发展。因此,HCC中SHh信号通路的有关研究对以细胞信号通路为靶标的特异性治疗策略具有重要价值。但目前为止,有关HCC的研究很少涉及在化疗药物作用下SHh信号通路所发生的变化。本论文以SHh通路激活的Hep3B细胞为研究材料,重点关注传统化疗药物5-FU对SHh信号通路活性的影响,以及该信号通路的活性改变是否与肝癌细胞的生长和迁移能力有关。本研究首先用MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide]法确认了以5-FU处理Hep3B细胞系48 h的半数抑制浓度为合适的工作浓度。5-FU作用于Hep3B细胞6 h,12 h,24 h和48 h时,通过半定量RT-PCR检测,发现5-FU可以下调SHh通路靶基因PTC1(Patched 1)和Glil(glioma-associated oncogene homolog 1)、配体基因SHH(Sonic Hedgehog)和受体基因SMO(Smoothened)的表达,表明5-FU对Hep3B细胞中SHh信号通路的主要元件的基因转录有影响。而在SHh通路无活性的HepG2细胞中,没有检测到PTC1和Glil表达,配体SHH也没有受到明显影响。为确定SHh通路的活性是否参与5-FU引发的生长阻滞和细胞凋亡,本研究通过pCS2-Glil质粒使Hep3B细胞过表达Glil,用MTT检测发现,当施加相同浓度的5-FU时,转染组比非转染组的Hep3B细胞具有更高的生长曲线;台盼蓝染色实验更是直接验证了MTT的检测结果;吖啶橙荧光染色实验发现,除了细胞核发生了明显的凝缩、边缘化和片段化,并有凋亡小体出现外,5-FU处理48h的转染组比非转染组的细胞凋亡比例明显减少;而用Hochest33258的荧光检测,除了发现细胞核在形态上有明显的凋亡特征外,还发现5-FU处理组比对照组的亮度强很多,荧光强度比较显著(*,p＜0.05),而Glil转染组的Hep3B细胞与对照组相比,虽然荧光强度有所增强,但程度不明显;用TUNEL检测以及相应的荧光强度的统计学分析发现,实验中Glil转染组中TUNEL阳染的细胞数量减少,而未转染Glil的实验组中阳染的细胞数量较多,5-FU引发的凋亡更为明显。本研究发现,和对照组以及5-FU处理的非转染组相比,转染组Hep3B细胞中Glil的过表达可以拮抗5-FU引发的细胞生长阻滞和细胞凋亡。使Hep3B细胞过表达Glil,从而上调SHh通路活性上调,可在一定程度上降低Hep3B细胞对5-FU的敏感性,增强Hep3B细胞对5-FU的拮抗作用。本研究的划痕损伤愈合实验发现,使Hep3B细胞过表达Glil可以拯救5-FU引起的迁移能力的降低,表明在SHh通路激活的肿瘤细胞中5-FU对细胞迁移能力的影响可能涉及对通路活性的抑制。进一步研究发现,过表达Glil的Hep3B细胞可以拮抗5-FU引发的PTC1蛋白亚细胞定位的改变。本研究用荧光免疫细胞化学检测法(ICC,Immunocytochemistry)对Hep3B细胞中PTC1蛋白的亚细胞定位的变化进行了检测,发现当用5-FU处理一定时间后,大多数PTC1蛋白分子失去了它们在膜上的定位,并在细胞质中散乱分布;随着5-FU作用时间的延长和细胞凋亡的进展,PTC1的信号越来越弱,分布也越来越散乱;5-FU处理48 h后,可观察到PTC1蛋白的表达基本消失,这个结果与前面Western Blot的结果是一致的。而使Hep3B细胞过表达Glil可以拮抗5-FU对PTC1蛋白亚细胞定位的改变。实验中还意外地发现PTC1在细胞有丝分裂期的独特分布形式,即分裂末期,胞内的PTC1蛋白似乎离开了它们在核膜附近的定位,大部分被募集到细胞膜上;尤其是在赤道板附近的即将内陷形成两个子细胞的膜区域,PTC1大量表达,暗示了PTC1可能参与有丝分裂过程的调控。本研究还对G1期晚期的细胞周期蛋白——cyclin E的表达进行了免疫细胞化学检测,发现5-FU作用于Hep3B细胞会引起cyclin E的显著下调;而在转染Glil的Hep3B细胞中,过表达的Glil可以抑制5-FU对cyclin E的下调。因此,过表达的Glil可以部分地抑制5-FU对Hep3B细胞的细胞周期阻滞作用。综上所述,本研究首次报道了5-FU引起的Hep3B细胞凋亡和迁移能力的抑制涉及SHh信号通路。使Hep3B细胞过表达Glil可以拮抗5-FU对Hep3B细胞的生长阻滞和细胞凋亡效应,拯救5-FU引起的Hep3B细胞的迁移能力的抑制、SHh信号通路受体PTC1的下调和分布变化以及细胞周期蛋白cyclin E的下调。此外,还意外发现在有丝分裂末期PTC1蛋白的分布特点,暗示了PTC1可能参与有丝分裂过程的调控。本研究为基于5-FU的治疗提供了新的治疗策略,即对于SHh信号通路激活的肝细胞癌患者,SHh信号通路抑制剂和化疗药物联合用药可能会代表一种更有前途的HCC治疗方法。这一结果为将来的治疗策略提供了新线索,而且可能在临床应用上具有重要意义。但仍需要进一步利用其他HCC细胞系研究以及进行体内研究。此外,本论文还研究了一种新的化疗试剂——四唑紫罗兰和传统的亚硝脲类化疗药物——盐酸尼莫斯汀(ACNU)的联合用药能引起化疗增效的现象。促使化疗增效的重要途径之一是提高肿瘤对传统临床化疗药物(如ACNU等)的敏感性,这已成为研究人员的重要课题。针对化疗药物不同的作用机制,增加肿瘤对化疗药物的敏感性的方式也不尽相同,但其目的都是为了提高化疗药物对肿瘤细胞的杀伤效果、减少对正常细胞的毒性。由于化疗药物的研究深度和选择范围有限,目前所采取的主要方式是通过药物联合作用来逆转各种的耐药机制。TV是一种能引发肿瘤细胞发生G1期阻滞和凋亡的四唑盐类化合物,近年来的研究表明,它可以和氮芥类烷化剂——BCNU联合作用,增加肿瘤细胞对卡氮芥的敏感性,因此是一种有希望的化疗试剂。然而TV和亚硝脲类烷化剂——ACNU的联合化疗,在治疗脑胶质瘤中是否起到增效作用,从而改善对脑胶质瘤联合化疗的疗效,目前尚未研究。因此,在较为理想的哺乳动物脑胶质瘤的体外模型——C6大鼠脑胶质瘤细胞中,本研究对TV和ACNU联合用药起到的增效作用及其可能的增效机制进行了研究,这可能对脑胶质瘤的联合化疗具有一定的意义。本研究表明,ACNU和TV联合用药可以提高每种药物单用对C6细胞的增殖抑制作用,促进细胞凋亡,起到增效作用;同时,这种增效作用会引起野生型p53蛋白,以及细胞周期相关蛋白p21、p27的显著上调;而且通过检测耐药相关蛋白——GSTs的酶活水平,本研究发现它和增效作用的相关性不高。因此,联合用药的增效机制仍将需要进一步深入探讨。由于本研究所涉及的对脑胶质瘤联合化疗的增效作用是一个多种因素参与的复杂过程,单方面考虑某一基因或蛋白的作用很难充分解释化疗中遇到的实际情况。因此,对脑胶质瘤的研究必须多基因、多途径地寻找其耐药机制,以逆转其耐药,增强化疗疗效。总之,本研究的两部分为提高化疗效果的基础性研究拓展了研究领域。虽然两部分的详细机制仍待探讨,但这些实验数据为深入研究肿瘤化疗作用机制及丰富化疗手段提供了有益的线索。创新点:首次报道了5-FU引起的Hep3B细胞凋亡和迁移能力的抑制涉及SHH信号通路活性的下调,为将来结合细胞信号通路改变的化疗增效提供思路;首次报道了TV和ACNU药物联用起到化疗增效作用,为临床上联合用药提供新的线索。更多还原

【Abstract】 To increase the therapeutic efficacy for tumors so as to achieve thorough therapy finally is one of essential targets of current biology and medical science.As one of the main approaches to tumor treatment,chemotherapy itself had some defaults which are determined by the mechanisms of chemotherapeutic agents themselvees, such as the deficiency of selectivity and general deleterious side effect for patients. Thus,the therapeutic efficacies are limited.The improvement of therapeutic methods and the exploration of new chemotherapeutic agents little side effect have been an essential task for researchers.The hot topics about current chemotherapy include following:one,to elevate the specificity and chemotherapeutic efficacy through switching on or off the activity of cell signaling pathway related to proliferation, apoptosis and metabolism by investigating and developing specific small compounds or molecules with biological activities,which is the most promising and potential approaches;two,to sensitize the tumor cells to chemicals(mainly through overcoming the drug-resistance of cells) by means of combination chemotherapy of traditional chemotherapeutic agents and new drugs(including newly-developed chemicals and natural drugs that had inhibitive effect of tumors),which is an important approach for better chemotherapeutic efficacy;three,to amend the medication ways by various means of targeting topical solid tumors directly and releasing slowly in order to inhibit tumors longer and kill the tumor cells.It is shown that some of signaling pathway of animal embryonic development correlated closely with tumorigenesis and tumor development,such as Wnt,Hh, Notch,BMP signaling pathways,etc.The relationship between Hh signaling pathway and tumors is one of hot spots in research in recent years.Hh signaling pathway is not only extremely important for the patterning and cell fate decision,but also crucial in tumorigenesis and tumor development.Recently,the role and significance of Hh signaling in human hepatocellular cancer(HCC) is highlighted by and by.It is demonstrated that SHh(Sonic Hedgehog) signaling pathway is frequently activated in HCC and participate in carcinogenesis and development of HCC.Thus,the correlated research on Hh signaling pathway has essential values in specific treatment strategy targeted by cell signaling pathway.By far,the research on HCC seldom involved the alteration of SHh signaling pathway with chemotherapeutic treatment.The research materials in present dissertation is based on Hep3B cells which has activated SHh signaling pathway,and what we cared is whether the alterations of Hh signaling activity has relationship with the growth and migration ability of HCC cells.The present study confirmed proper work concentration by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay,which is 50% inhibition concentration for Hep3B cells treated for 48 h.When treatment elapsed by 6 h,12 h,24 h and 48 h,the results from semi-quantitative RT-PCR found the down regulation of target genes PTC1(Patched 1) and Gli1(glioma-associated oncogene homolog 1),ligand gene SHH(Sonic Hedgehog) and receptor gene SMO (Smoothened),indicated the influence of 5-FU on main components in SHh signaling pathway.While in the case of HepG2 cells,there is no expression of PTC1 and Gli1,and the expression of SHH ligand was not affected obviously.To confirm whether the cell growth arrest and apoptosis induced by 5-FU involves the activity of SHh signaling pathway,the present study made Gli1 overexpressed in Hep3B cells and when exposed to the same concentration of 5-FU, the transfection groups had higher survival curve than non-transfection groups in MTT assay.The results in MTT assay was also demonstrated directly by Trypan Blue assay;it is observed under fluorescent microscope in acridine dyeing assay that the nuclei in part of Hep3B cells happened to be of gragmentation,marginalization and chromatic agglutination with apoptotic body scattered around,and besides these, it was found that 5-FU treated transfection groups at 48 h had significant decrease comparing with non-transfection groups.As for Hochest33258 fluorescent assay, except for obvious apoptotic characteristics of nuclei,we found the average fluorescent intensity in 5-FU treated groups was much stronger than control groups (~*,p＜0.05),while the average fluorescent intensity in transfection groups is also stronger than that in control groups,but with no significant difference.TUNEL assay and corresponding fluorescent intensity assay with statistical analysis showed remarkable decrease trend in transfection groups with 5-FU treated,comparing with non-transfection groups with 5-FU treated.So,in present study the overexpression of Gli1 in trasfection group was found to antagonize the cell growth arrest and apoptosis induced by 5-FU.The overexpression of Gli1 in Hep3B cells can also elevate the activity of SHh signaling pathway,thus it decreased the sensitivity of Hep3B cells with 5-FU treatment and increased antagonistic effect in Hep3B cells.Our study found through scratch wound closure assay showed that the overexpression of Gli1 can rescue the decrease in motility of Hep3B cells induced by 5-FU,and this indicated that 5-FU may have influenced motility of SHh-activated tumor cells,which is involved inhibition of the activity of pathway.Besides,the overexpression of Gli1 in Hep3B cells may antagonize the change of subcellular location of PTC1.In this study,we detected the subcellular location of PTC1 in Hep3B cells through immunocytochemistry(ICC) assay,and found most of the PTC1 lost their location on membrane and distributed dispersedly with 5-FU treated for certain period.With time passed by and apoptosis progression,the signal from PTC1 became weaker and distribution became more scattered.After the treatment for 48 h,the expression of PTC1 was almost disappeared,this is consistent with the results in Western Blot.What’s more,the overexpression of Gli1 may antagonize the alteration of subcellular location for PTC1 in Hep3B cells treated with 5-FU.Unexpectedly,the unique intracellular distribution of PTC1 in telophase of mitosis was found in our study,which possibly meant the isolation of PTC1 from their location near nuclear envelope and the recruitment of a major of PTC1 on cell membrane.Especially,near the equatorial plate region where the membrane was going to invaginate to form daughter cells,PTC1 was expressed in large amount,and this implies that PTC1 may involve the regulation of mitosis.Furthermore,the study detected the expression of cyclin E,the cyclin that can function in late G1 phase,in ICC assay.We found significant down regulation of cyclin E induced by 5-FU in Hep3B cells.While in transfection groups,the overexpression of Gli1 in Hep3B cells can inhibit the down regulation of cyclin E. Thus,the overexpression of Gli1 may partially inhibit the cell growth arrest of Hep3B.In conclusion,our study for the first time reported that down-regulation of SHh signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells.The overexpression of Gli1 can antagonize the effect of the cell growth arrest and apoptosis,and can also rescue motility inhibition of Hep3B cells induced by 5-FU,down regulation of receptor PTC1 with distribution change and cell cycle related protein,cyclin E.Besides,we found a unique distribution character of PTC1 at telophase in mitosis unexpectedly,which implies its involvement in regulation of mitosis.The study provides a new therapeutic strategy for 5-FU based therapy.As for HCC patients with SHh signaling pathway activated,the combination of inhibitor of SHh signaling pathway and other chemotherapeutic agents may represent a more promising therapy for HCC patients.In addition,the study made the research on combination chemotherapy of a new chemical,TV(tetrazolium violet) and traditional nitrosourea chemotherapeutic agents,ACNU(nimustine hydrochloride),which can cause synergistic effect in combination chemotherapy.One of important ways to promote synergy in chemotherapy is to increase the sensitivity of therapeutic agents,especially for traditional clinical drugs,such as ACNU.And this have become to important topics for researchers.Targeting to different mechanisms of chemotherapeutic agents,researchers have increased the sensitivity of different therapeutic agents with different ways,but all the destinations are to increase the chemotherapeutic effect of drugs or decrease their deleterious effect to normal cells.For the sake of limited research depth and selective range,in present,the common way to reverse various possible drug-resistance mechanisms is combination chemotherapy.TV is a tetrazolium salts that can elicit cell growth arrest at G1 phase and apoptosis for tumor cells.Recent research indicated that TV can cooperate with BCNU(carmustine) to elevate the sensitivity of tumor ceils to BCNU. Therefore,TV is a promising chemotherapeutic agent.But whether the combination chemotherapy of ACNU and TV can cause synergy in treatment for gliomas,and whether this combination chemotherapeutic approaches for gliomas will be improved is unknown.Thus,we perform the tests on C6 rat glioma cells which is an ideal model in vitro for gliomas in mammals and we investigate the synergistic effect of this combination chemotherapy and possible synergistic mechanism,which may have essential significancy in therapy in gliomas.The present study indicated that the combination chemotherapy of ACNU and TV can promote proliferative inhibition of in C6 cells and promote apoptosis, comparing with treatment in single drug;at the same time,this combination chemotherapy will cause up regulation in wild type p53 protein,cell cycle related protein p21 and p27;furthermore,we measured the activity of drug-resistant protein, only to find little relevance.Thus,the synergistic mechanism of this combination needs further tests and other analysis in future.Since the synergistic effect of combination chemotherapy for gliomas is a complicated process with multiple factors,it is hard to elucidate the practical problems with the function of only one gene or one protein.Thus,the research on gliomas must seek the drug-resistant mechanisms from multiple genes and approaches in order to reverse the drug-resistance and improve the chemotherapeutic efficacy.On the whole,the two parts of this study improved basic research on chemotherapeutic efficacy and expand the investigation field.Although the specific mechanisms is yet required to further investigation,these data provide the helpful clues to further investigation on chemotherapeutic mechanism of action,and to enrich the chemotherapeutic approaches.Innovations:To report for the first time that down-regulation of SHh signaling pathway activity is involved in 5-fluorouracil-induced apoptosis and motility inhibition in Hep3B cells,which provides new clues to chemotherapeutic synergy in future, combined with alterations of cell signaling pathways.To report for the first time that combination chemotherapeutic of TV and ACNU will bring about synergistic effect on C6 cells,which will provide effective scheme to combination chemotherapy clinically.更多还原