IL-13Ra2抗原肽致敏树突状细胞对人脑胶质瘤及其肿瘤干细胞的杀伤效应研究
|
摘要
目的胶质瘤是中枢神经系统(central nervous system, CNS)最常见的恶性肿瘤,起源于神经胶质细胞,包括星形细胞系瘤,少突胶质细胞系肿瘤,混合性胶质细胞瘤,室管膜瘤等。据统计,胶质瘤约占颅内原发性肿瘤的35.26-60.96%(平均44.69%)。由于肿瘤分化不全呈侵袭性生长,手术切除不能够彻底,预后较差。即使进行放化疗也都很难有效根除,病人平均生存期约14.6个月,一般不到两年,极大威胁了人们的生命健康。因此,寻找新的胶质瘤治疗方式成为近20年来研究的热点。随着对中枢神经系统及胶质瘤免疫特性认识的逐渐深入,生物免疫治疗已成为第四种治疗模式,是治疗颅内肿瘤最有前景的新疗法。DC疫苗由于安全,有效及低毒,是胶质瘤免疫治疗的重点。树突状细胞(dendritic cells,DCs)是目前发现的功能最强大的专职抗原呈递细胞,形态上具有树突状结构,主要组织相容性复合体及共刺激分子如CD86、CD80等的表达水平较高,能够有效摄取并行抗原处理,将之递呈与T淋巴细胞,从而具有启动作用,激发初次细胞免疫应答,引发一系列免疫反应。研究表明,多种方法制备的DC有一定的抗瘤效果。目前致敏DC的抗原有纯化的蛋白质、抗原肽、肿瘤细胞裂解物、肿瘤DNA或RNA等。由于胶质瘤抗原性较弱,且不同个体的胶质瘤间存在异质性,目前研究尚未发现其特异性公共抗原,使DC疫苗目前只能够行个体化治疗,无法实现疫苗的公共化。找到一种在胶质瘤中表达较为广泛的特异性蛋白成为目前脑胶质瘤疫苗研究急需解决的问题。IL-13Ra2是由380个氨基酸残基组成的膜受体,编码基因定位于xq24,属红细胞生成素受体家族。近年来,研究发现IL-13Ra2其在恶性肿瘤细胞尤其是胶质瘤中存在特异性高表达,且恶性程度越高其表达越强,而在正常组织及器官中几乎不表达,由此指出IL-13Ra2是恶性肿瘤的一种受体蛋白,作为一种可能的胶质瘤特异性标志物,有可能在胶质瘤的诊断及治疗方面做出贡献。国外研究已发现IL-13Ra2具有明显的免疫原性及抗原性,可应用于免疫疗法针对恶性胶质瘤细胞,在抗胶质瘤研究中极具潜力,使用IL-13Ra2蛋白致敏的DC疫苗可能同样具有杀伤肿瘤细胞的效果,为DC疫苗的公共化做出贡献。
近年来研究发现胶质瘤中存在少部分具干细胞特征的细胞,其具备以下几种特点：无限增殖及自我更新能力、多向分化潜能及高致瘤性、表达干细胞特异性标记。该细胞被称为肿瘤干细胞,高表达多种抗凋亡基因及耐药基因,多处于细胞周期的相对静止期,对放化疗不敏感,不易杀灭。Ignatova发现该类细胞表达CD133及Nestin, Singh等则发现,小鼠颅内接种100个CD133+细胞即可成瘤。胶质瘤干细胞是肿瘤复发的根源。随肿瘤干细胞研究的深入,其作为DC抗原致敏物致敏DC疫苗,诱导出的T细胞抗肿瘤免疫反应明显强于传统DC制备方法得到的疫苗,证明肿瘤干细胞在免疫治疗中具有抗原性,为免疫治疗研究打开了新的视角。由于肿瘤干细胞是肿瘤复发的根源,针对肿瘤干细胞的免疫治疗为根治胶质瘤带来希望。
本实验通过检测IL-13Ra2蛋白在脑胶质瘤及正常脑组织中的表达情况,使用IL-13Ra2抗原肽及胶质瘤干细胞的细胞裂解物致敏异体同血型树突状细胞制备DC疫苗,刺激T淋巴细胞,观察是否能有效的杀伤胶质瘤细胞,并与传统胶质瘤全细胞抗原致敏的DC疫苗效果相比较,从而探讨IL-13Ra2蛋白及肿瘤干细胞在胶质瘤免疫治疗中的重要性。采用统计学软件SPSS17.0进行数据统计学分析。
方法
本实验分为三部分：
第一部分IL-13Ra2蛋白在脑胶质瘤及U251细胞中的表达：收集取得的手术标本制成组织蜡块,u251细胞复苏培养制成细胞爬片,采用免疫组化SABC法(细胞免疫组化及组织免疫组化)检测59例脑胶质瘤5例正常脑组织及U251细胞系中IL-13Ra2蛋白的表达,并对其与肿瘤恶性程度的关系进行统计学分析。
第二部分胶质瘤细胞及肿瘤干细胞的培养：收集术中取得的胶质瘤标本,剪切消化,在含血清培养基(DMEM高糖+10%人体血浆)条件下行胶质瘤细胞原代培养；原代胶质瘤细胞以无血清培养法在干细胞培养基(DMEM/F12+20ng/ml bFGF+20ng/ml EGF)条件下培养原代胶质瘤干细胞,生成肿瘤球后行肿瘤干细胞分化实验及流式细胞术检测CD133阳性细胞的表达率。U251细胞系细胞复苏及细胞培养(DMEM高糖+10%FBS)。第三部分：DC诱导制备及DC疫苗诱导的细胞杀伤效应检测：取正常人外周血,淋巴分离液分离,离心机离心后吸取单个核细胞以1640培养基+10%人体血浆37。C,5%CO2的恒温培养箱内培养培养2小时,收集非粘附细胞后用含有粒-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)的培养基诱导培养,培养过程中倒置显微镜下观察形态,流式细胞仪检测DC表面标记物CD14、CD83、CD80、CDla、CD86及HLA-DR。取实验第二部分培养的原代胶质瘤细胞,胶质瘤干细胞及U251细胞,反复冻融裂解后离心,制备全细胞抗原。将IL-13Ra2蛋白及上述所得肿瘤全细胞抗原及肿瘤干细胞抗原分别与成熟DC混合,培养箱中培养24小时,获得不同DC疫苗。用人T细胞富集液孵育同源外周血后以淋巴细胞分离液分离并离心,取得T淋巴细胞,T淋巴细胞与DC疫苗共培养后行T细胞增殖实验及T细胞杀伤实验,比较不同DC疫苗的肿瘤的杀伤效果。统计学分析：结果以均值土标准差表示,多组分析采用单因素方差分析,两两比较用t检验。P<0.05为差异有统计学意义。
结果IL-13Ra2不在正常的脑组织中表达,而59例胶质瘤组织中有33例IL-13Ra2表达阳性(阳性率55.9%),差异显著。胶质瘤中IL-13Ra2的表达在高度恶性胶质瘤与低级别胶质瘤之间的差异有统计学意义。U251细胞系IL-13Ra2表达阳性。U251细胞及胶质瘤培养成功,在含血清培养基中显微镜下可见其细胞呈梭形,部分呈星形及多角形,胞质透亮,贴壁单层生长。生长迅速,约1周传代一次。4代后可见细胞突起较多,多角形占大部分。无血清干细胞培养基内培养1周多悬浮生长,形成悬浮肿瘤克隆球,折光性较好,流式检测细胞标记CD133阳性,当接种于含血清培养基后肿瘤细胞球逐渐贴壁,细胞逐渐迁出,贴壁分化的细胞形状与原代培养肿瘤细胞相似。在细胞因子GM-CSF和IL-4作用下可成功从人外周血单核细胞中诱导出DC,相差显微镜镜下可见单核细胞贴壁生长,悬浮细胞逐渐增多,体积增大不规则,有典型树突状形态突起,符合成熟形态。流式细胞仪检测可见PBMC-标志物CD14低表达,DCS表面标志物表达率增加,(特征性表面标记CD8387.1%、CDla81%,高表达功能相关抗原CD8078%、CD8684.2%及HLA-DR83%)提示人外周血中诱导到大量高纯度成熟DC。细胞体外杀伤实验结果表明,与单纯DC组相比,IL-13Ra2,肿瘤全细胞抗原及肿瘤干细胞抗原致敏的DC细胞均能明显刺激T细胞增殖,有效杀灭胶质瘤细胞。相比其他实验组,肿瘤干细胞抗原致敏的DC疫苗活化的CTL胶质瘤的杀伤效应更强(P<0.05), IL-13Ra2致敏DC组与肿瘤全细胞抗原致敏DC组相比效果无明显差异(P>0.05),在以脑肿瘤干细胞为靶细胞的体外杀伤实验中干细胞抗原裂解物对肿瘤细胞的杀伤力最强,与IL-13Ra2致敏DC组及单纯DC细胞组相比差异有显著的统计学意义(P<0.001)。而IL-13Ra2致敏DC组杀伤率较低,与单纯DC组相比无统计学意义。(P>0.05)。DC与T细胞比例1：10时刺激能力及杀伤活性最强。
结论IL-13Ra2蛋白在正常脑组织中不表达,而在胶质瘤及U251细胞系中存在广泛表达,且表达与恶性程度有关,是一种理想的胶质瘤免疫治疗靶标；原代胶质瘤组织及U251细胞系在无血清培养条件下可成功培养出足够数量的胶质瘤干细胞；人类外周血来源单个核细胞在体外GM-CSF和IL-4联合诱导培养下可获得充足的成熟DC树突状细胞,为DC的肿瘤免疫治疗提供了细胞来源。肿瘤干细胞抗原致敏的DC疫苗能有效刺激抗原特异性CTL,杀伤胶质瘤细胞功能较IL-13Ra2致敏DC疫苗及肿瘤全细胞抗原致敏DC更强。而IL-13Ra2致敏DC疫苗与肿瘤全细胞抗原致敏DC疫苗也有明显的胶质瘤细胞杀伤效果,有潜在临床运用价值。
Objective Glioma is the most common malignant tumor originated from glial cells in central nervous system (CNS),which including strocytoma, mesoglioma, admixed spongiocytoma and ependymoma. According to statistics, the percentage of glioma in primary intracranial tumors range from35.26percent to60.96percent. Due to the. infiltrative growth, the surgical removal of glioma were incomplete, surgical resection combined with radiotherapy and chemotherapy have same result. Mostly patients resulted in poor outcomes. The mean survival time of glioma was14.6months approximately, usually less than two years. The situation becoming today's health menace, so looking for a new therapy become more and more important in the past20years.As the realization of central nervous system and immune characteristic began to sink in, biological immunotherapy has become the fourth therapeutic mode of glioma, which seems like the most promising options for future cancer treatment. Due to the features like safe, effective and low toxicity, DC vaccine becoming the focus of glioma immunotherapy. The dendritic cells (DC) is known as the most powerful antigen presenting cell in the body, which have dendritic structure and High expression levels of the major histocompatibility complex and costimulatory molecules as CD80, CD86. It can uptake and processing antigens, present them to T lymphocytes effectively, triggering a series of immune response. The study found that the DC vaccines made by different methods have certain anti-tumor effect.DC can be sensitization by antigen as purified protein, antigenic peptide, lysates of tumor Cells, and DNA or RNA, Because glioma has low antigenicity and different individuals heterogeneity, we not yet found specific antigen, the DC vaccine was only used in individualized treatment.so,find a antigen which expressed extensively in glioma become urgent problem to be solved. Interleukin-13receptor alpha2(IL-13Ra2) is a membrane receptor composed of380amino acid residues, belong to the erythropoietin receptor family. Its encoding gene is located on Xq24.In recent years, studies have found that IL-13Ra2have highly tissue-specific expression in malignant rumor,especially in gliomas. The higher malignancy glioma has, the stronger it express, while almost no expression in normal tissues and organs. So as a possible glioma-specific marker, IL-13Ra2may make a contribution to the diagnosis and treatment of glioma in future.research abroad have found IL-13Ra2showed the expected antigenicity and immunogenicity, that's big potential for future cancer treatment. DC sensitized by IL-13Ra2also have certain anti-tumor effect, and this fruit would be contributed to the public of DC vaccine. Today we found there are mall deposits of cells with stem cell characteristics in glioma, have characteristics like Unlimited proliferation, self-renewal, multiple differentiation potential and tumorigenicity. This kind of cells express specific markers of stem cells.The cells are called for cancer stem cells, who have high expression of some anti-apoptotic genes and drug resistance genes.most of them stay in relative resting stage of the cell cycle,so it is't sensitive to the chemotherapy,and difficult to perish.Ignatova found they expressing CD133and Nestin,and Singh found when mice challenged with100CD133+cells intracranial induced tumor.So, tumor stem cells are the source of tumor recurrence.As the research of tumor stem cells began to sink in, DC vaccine which used tumor stem cell as antigen have induced stronger immune response than traditional DC vaccines, proved that tumor stem cells own antigenicity duing the treatment,offer exciting new insights into immunotherapy. In this study, we detect the expression of IL-13Ra2in glioma and normal brain tissue, observe the ability to trigger immune response of DC vaccines which sensitized with IL-13Ra2, glioma cells and brain tumor stem cells, and compare with traditional DC vaccines, Discuss the importance of IL-13Ra2and brain tumor stem cell in immunotherapy.
Methods The experiment was divided into four parts; Part one, Expression of IL-13Ra2in human glioma and U251cell line:Collect tissues duing operation to get paraffin blocks,resuscitate and culture U251cells to let them growing on glass coverslips The SABC immunohistochemical staining method was used to examine the protein expression of IL-13Ra2in59gliomas,5normal brain tissues and U251cells. The correlations of their expression of IL-13Ra2with malignant degrees was analyzed statistically. Part two, Culture of Glioma cell and brain tumor stem cell and preparation of cellular antigen: Collect the glioma tissues during surgery,After digested and dissoeiated, use DMEM medium containing10%Human plasma to cultured Primary glioma cells. Then cultured the cells in serum-free medium (DMEM/F12+20ng/mLbFGF,+20ng/mLEGF). cell differentiation experiment have done when spheres formed, and use flow cytometry to measured the rate of CD133+cells in the brain tumor stem cells, use DMEM medium containing10%FBS to resurrected and cultured U251cell line. Preparation of DC and DC vaccine, and Vitro cytotoxicity experiment induced to the glioma:Taken normal human peripheral blood, lymphatic separation medium, centrifuged after1640+10%autologous plasma absorb mononuclear cells cultured in37°C,5%CO2incubator for2hours, separated human peripheral blood mononeuclear cells(PBMC) from Human PeriPheral blood, then cultured the cells in1640medium containing10%for2hours.then collected and induced the non-adherent cells in1640medium containing10%Human plasma,add GM-CSF and IL-4. observed cells with light microscope, and use flow cytometry to measured their surface markers。Mixed mature DC with IL-13Ra2protein,tumor cell antigen and a tumor stem cell antigen, then cultured them for24hours to get DC vaccines. Isolated T cells from human peripheral blood mononeuclear cells (PBMC),then cultured T lymphocytes and DC in the RPMI1640containing10%Human plasma, compared anti-tumor effect with different DC vaccines.Statistical analysis:Results are expressed as mean±SD. The t test was used for comparisons of two groups,oneway ANOVA was used for Comparisons of multiple groups. Difference is considered significant at P<0.05
Results There was no expression of IL-13Ra2in all cases of normal brain tissues. However,33of the59glioma tissues showed positive expression of IL-13Ra2(55.9%), There were specially significant difference between them(P<0.01).duing the glioma group, the expression of IL-13Ra2was significantly different between low grade glioma (I-II grade) and high grade glioma (Ⅲ桰Vgrade)(P<0.01)。 U251cell line also showed positive expression of IL-13Ra2too. We successfully achieved enough U251cells and glioma cells by culturing them in serum containing medium, the cells seen under the microscope have shapes as spindle style, star style and polygonal style. The cell have translucent endochytema, and grown in the monolayer and adherence way on the bottle. It grows rapidly, passage in every week. After4th generation,we found the polygonal shape became the most shape in the cells. Eulturing glioma cells in serum-free medium we found BTSC grown as The cloning balls of cells,4th generation protuberance more visible, accounted for most of the polygonal shape. Serum-free stem cell cultured culture medium more than1week grown in suspension, formed the the suspensions tumor clone ball, the refractive better, flow cytometry cell markers CD133positive for expression rates of the different patients. flow cytometry found many CD133+cells in the brain tumor stem cells, Expression rate of low grade gliomas became higher with culture time increased, when cells was inoculated into serum containing medium, many cells move out of clone ball,The characterization of them was same as primary glioma cells。DCs could be isolated from PBMC by GM-CSF and IL-4. under the microscope we can seen the typical dendritic structure on DC's membrane surface.Flow cytometry detected the PBMCs surface marker, CD14, have low expression on DCs,while the expression rates of DCS characteristic surface markers higher than before,(CD8387.1%, CDla81%, CD8684.2%、 HLA-DR83%、CD8078%), we get large number of purity mature DC from PBMC.The Vitro Cells Cytotoxic experiment results show that, Compared with pure DC group, DC vaccines loaded by IL-13Ra2, glioma cell antigen and brain tumor stem cell antigen could significantly stimulated the proliferation of T lymphocytes, and abolish glioma cells Effectively. Compared to other groups, DC vaccines loaded by brain tumor stem cell antigen have the strongest effect to activated CTL (P<0.05), while IL-13Ra2group and tumor cell antigen group have no significant difference in the effect (P>0.05). IL-13Ra2group have weakly effect to induced CTL for glioma stem cell, when compare it with the pure DC group,we got no statistical significance from statistical analysis (P>0.05). The stimulation index of used brain tumor stem cell antigen was higher than others in the same stimulator to responder ratio, and when the ratio was1:10,the stimulation index was the highest one.
Conclusion:IL-13Ra2protein do not express in normal brain tissue,but widely expressed in glioma and U251cell line.The Expression of IL-13Ra2is associated with the malignant degrees of glioma, so it is an ideal immunotherapy target; We can get enough brain tumor stem cells in the way of culture Glioma cell and U251cell line in serum-free medium; Sour ce of of human peripheral blood mononuclear cells from in vitro GM-CSF and IL-4induce--d cultured available under plenty of mature DC dendritic cells provide a source of cells for tumor immunotherapy in DC vaccines. Compared to IL-13Ra2group and glioma cell antig-en, DC vaccines loaded by brain tumor stem cell antigen have the strongest effect to active-ted CTL which directed against glioma cell and brain tumor stem cell, besides, DC va-ccine loaded by IL-13Ra2and glioma cell antigen also have obviously effect to kill glio-mas cells,so they also have has potential value in clinical therapy.
近年来研究发现胶质瘤中存在少部分具干细胞特征的细胞,其具备以下几种特点：无限增殖及自我更新能力、多向分化潜能及高致瘤性、表达干细胞特异性标记。该细胞被称为肿瘤干细胞,高表达多种抗凋亡基因及耐药基因,多处于细胞周期的相对静止期,对放化疗不敏感,不易杀灭。Ignatova发现该类细胞表达CD133及Nestin, Singh等则发现,小鼠颅内接种100个CD133+细胞即可成瘤。胶质瘤干细胞是肿瘤复发的根源。随肿瘤干细胞研究的深入,其作为DC抗原致敏物致敏DC疫苗,诱导出的T细胞抗肿瘤免疫反应明显强于传统DC制备方法得到的疫苗,证明肿瘤干细胞在免疫治疗中具有抗原性,为免疫治疗研究打开了新的视角。由于肿瘤干细胞是肿瘤复发的根源,针对肿瘤干细胞的免疫治疗为根治胶质瘤带来希望。
本实验通过检测IL-13Ra2蛋白在脑胶质瘤及正常脑组织中的表达情况,使用IL-13Ra2抗原肽及胶质瘤干细胞的细胞裂解物致敏异体同血型树突状细胞制备DC疫苗,刺激T淋巴细胞,观察是否能有效的杀伤胶质瘤细胞,并与传统胶质瘤全细胞抗原致敏的DC疫苗效果相比较,从而探讨IL-13Ra2蛋白及肿瘤干细胞在胶质瘤免疫治疗中的重要性。采用统计学软件SPSS17.0进行数据统计学分析。
方法
本实验分为三部分：
第一部分IL-13Ra2蛋白在脑胶质瘤及U251细胞中的表达：收集取得的手术标本制成组织蜡块,u251细胞复苏培养制成细胞爬片,采用免疫组化SABC法(细胞免疫组化及组织免疫组化)检测59例脑胶质瘤5例正常脑组织及U251细胞系中IL-13Ra2蛋白的表达,并对其与肿瘤恶性程度的关系进行统计学分析。
第二部分胶质瘤细胞及肿瘤干细胞的培养：收集术中取得的胶质瘤标本,剪切消化,在含血清培养基(DMEM高糖+10%人体血浆)条件下行胶质瘤细胞原代培养；原代胶质瘤细胞以无血清培养法在干细胞培养基(DMEM/F12+20ng/ml bFGF+20ng/ml EGF)条件下培养原代胶质瘤干细胞,生成肿瘤球后行肿瘤干细胞分化实验及流式细胞术检测CD133阳性细胞的表达率。U251细胞系细胞复苏及细胞培养(DMEM高糖+10%FBS)。第三部分：DC诱导制备及DC疫苗诱导的细胞杀伤效应检测：取正常人外周血,淋巴分离液分离,离心机离心后吸取单个核细胞以1640培养基+10%人体血浆37。C,5%CO2的恒温培养箱内培养培养2小时,收集非粘附细胞后用含有粒-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)的培养基诱导培养,培养过程中倒置显微镜下观察形态,流式细胞仪检测DC表面标记物CD14、CD83、CD80、CDla、CD86及HLA-DR。取实验第二部分培养的原代胶质瘤细胞,胶质瘤干细胞及U251细胞,反复冻融裂解后离心,制备全细胞抗原。将IL-13Ra2蛋白及上述所得肿瘤全细胞抗原及肿瘤干细胞抗原分别与成熟DC混合,培养箱中培养24小时,获得不同DC疫苗。用人T细胞富集液孵育同源外周血后以淋巴细胞分离液分离并离心,取得T淋巴细胞,T淋巴细胞与DC疫苗共培养后行T细胞增殖实验及T细胞杀伤实验,比较不同DC疫苗的肿瘤的杀伤效果。统计学分析：结果以均值土标准差表示,多组分析采用单因素方差分析,两两比较用t检验。P<0.05为差异有统计学意义。
结果IL-13Ra2不在正常的脑组织中表达,而59例胶质瘤组织中有33例IL-13Ra2表达阳性(阳性率55.9%),差异显著。胶质瘤中IL-13Ra2的表达在高度恶性胶质瘤与低级别胶质瘤之间的差异有统计学意义。U251细胞系IL-13Ra2表达阳性。U251细胞及胶质瘤培养成功,在含血清培养基中显微镜下可见其细胞呈梭形,部分呈星形及多角形,胞质透亮,贴壁单层生长。生长迅速,约1周传代一次。4代后可见细胞突起较多,多角形占大部分。无血清干细胞培养基内培养1周多悬浮生长,形成悬浮肿瘤克隆球,折光性较好,流式检测细胞标记CD133阳性,当接种于含血清培养基后肿瘤细胞球逐渐贴壁,细胞逐渐迁出,贴壁分化的细胞形状与原代培养肿瘤细胞相似。在细胞因子GM-CSF和IL-4作用下可成功从人外周血单核细胞中诱导出DC,相差显微镜镜下可见单核细胞贴壁生长,悬浮细胞逐渐增多,体积增大不规则,有典型树突状形态突起,符合成熟形态。流式细胞仪检测可见PBMC-标志物CD14低表达,DCS表面标志物表达率增加,(特征性表面标记CD8387.1%、CDla81%,高表达功能相关抗原CD8078%、CD8684.2%及HLA-DR83%)提示人外周血中诱导到大量高纯度成熟DC。细胞体外杀伤实验结果表明,与单纯DC组相比,IL-13Ra2,肿瘤全细胞抗原及肿瘤干细胞抗原致敏的DC细胞均能明显刺激T细胞增殖,有效杀灭胶质瘤细胞。相比其他实验组,肿瘤干细胞抗原致敏的DC疫苗活化的CTL胶质瘤的杀伤效应更强(P<0.05), IL-13Ra2致敏DC组与肿瘤全细胞抗原致敏DC组相比效果无明显差异(P>0.05),在以脑肿瘤干细胞为靶细胞的体外杀伤实验中干细胞抗原裂解物对肿瘤细胞的杀伤力最强,与IL-13Ra2致敏DC组及单纯DC细胞组相比差异有显著的统计学意义(P<0.001)。而IL-13Ra2致敏DC组杀伤率较低,与单纯DC组相比无统计学意义。(P>0.05)。DC与T细胞比例1：10时刺激能力及杀伤活性最强。
结论IL-13Ra2蛋白在正常脑组织中不表达,而在胶质瘤及U251细胞系中存在广泛表达,且表达与恶性程度有关,是一种理想的胶质瘤免疫治疗靶标；原代胶质瘤组织及U251细胞系在无血清培养条件下可成功培养出足够数量的胶质瘤干细胞；人类外周血来源单个核细胞在体外GM-CSF和IL-4联合诱导培养下可获得充足的成熟DC树突状细胞,为DC的肿瘤免疫治疗提供了细胞来源。肿瘤干细胞抗原致敏的DC疫苗能有效刺激抗原特异性CTL,杀伤胶质瘤细胞功能较IL-13Ra2致敏DC疫苗及肿瘤全细胞抗原致敏DC更强。而IL-13Ra2致敏DC疫苗与肿瘤全细胞抗原致敏DC疫苗也有明显的胶质瘤细胞杀伤效果,有潜在临床运用价值。
Objective Glioma is the most common malignant tumor originated from glial cells in central nervous system (CNS),which including strocytoma, mesoglioma, admixed spongiocytoma and ependymoma. According to statistics, the percentage of glioma in primary intracranial tumors range from35.26percent to60.96percent. Due to the. infiltrative growth, the surgical removal of glioma were incomplete, surgical resection combined with radiotherapy and chemotherapy have same result. Mostly patients resulted in poor outcomes. The mean survival time of glioma was14.6months approximately, usually less than two years. The situation becoming today's health menace, so looking for a new therapy become more and more important in the past20years.As the realization of central nervous system and immune characteristic began to sink in, biological immunotherapy has become the fourth therapeutic mode of glioma, which seems like the most promising options for future cancer treatment. Due to the features like safe, effective and low toxicity, DC vaccine becoming the focus of glioma immunotherapy. The dendritic cells (DC) is known as the most powerful antigen presenting cell in the body, which have dendritic structure and High expression levels of the major histocompatibility complex and costimulatory molecules as CD80, CD86. It can uptake and processing antigens, present them to T lymphocytes effectively, triggering a series of immune response. The study found that the DC vaccines made by different methods have certain anti-tumor effect.DC can be sensitization by antigen as purified protein, antigenic peptide, lysates of tumor Cells, and DNA or RNA, Because glioma has low antigenicity and different individuals heterogeneity, we not yet found specific antigen, the DC vaccine was only used in individualized treatment.so,find a antigen which expressed extensively in glioma become urgent problem to be solved. Interleukin-13receptor alpha2(IL-13Ra2) is a membrane receptor composed of380amino acid residues, belong to the erythropoietin receptor family. Its encoding gene is located on Xq24.In recent years, studies have found that IL-13Ra2have highly tissue-specific expression in malignant rumor,especially in gliomas. The higher malignancy glioma has, the stronger it express, while almost no expression in normal tissues and organs. So as a possible glioma-specific marker, IL-13Ra2may make a contribution to the diagnosis and treatment of glioma in future.research abroad have found IL-13Ra2showed the expected antigenicity and immunogenicity, that's big potential for future cancer treatment. DC sensitized by IL-13Ra2also have certain anti-tumor effect, and this fruit would be contributed to the public of DC vaccine. Today we found there are mall deposits of cells with stem cell characteristics in glioma, have characteristics like Unlimited proliferation, self-renewal, multiple differentiation potential and tumorigenicity. This kind of cells express specific markers of stem cells.The cells are called for cancer stem cells, who have high expression of some anti-apoptotic genes and drug resistance genes.most of them stay in relative resting stage of the cell cycle,so it is't sensitive to the chemotherapy,and difficult to perish.Ignatova found they expressing CD133and Nestin,and Singh found when mice challenged with100CD133+cells intracranial induced tumor.So, tumor stem cells are the source of tumor recurrence.As the research of tumor stem cells began to sink in, DC vaccine which used tumor stem cell as antigen have induced stronger immune response than traditional DC vaccines, proved that tumor stem cells own antigenicity duing the treatment,offer exciting new insights into immunotherapy. In this study, we detect the expression of IL-13Ra2in glioma and normal brain tissue, observe the ability to trigger immune response of DC vaccines which sensitized with IL-13Ra2, glioma cells and brain tumor stem cells, and compare with traditional DC vaccines, Discuss the importance of IL-13Ra2and brain tumor stem cell in immunotherapy.
Methods The experiment was divided into four parts; Part one, Expression of IL-13Ra2in human glioma and U251cell line:Collect tissues duing operation to get paraffin blocks,resuscitate and culture U251cells to let them growing on glass coverslips The SABC immunohistochemical staining method was used to examine the protein expression of IL-13Ra2in59gliomas,5normal brain tissues and U251cells. The correlations of their expression of IL-13Ra2with malignant degrees was analyzed statistically. Part two, Culture of Glioma cell and brain tumor stem cell and preparation of cellular antigen: Collect the glioma tissues during surgery,After digested and dissoeiated, use DMEM medium containing10%Human plasma to cultured Primary glioma cells. Then cultured the cells in serum-free medium (DMEM/F12+20ng/mLbFGF,+20ng/mLEGF). cell differentiation experiment have done when spheres formed, and use flow cytometry to measured the rate of CD133+cells in the brain tumor stem cells, use DMEM medium containing10%FBS to resurrected and cultured U251cell line. Preparation of DC and DC vaccine, and Vitro cytotoxicity experiment induced to the glioma:Taken normal human peripheral blood, lymphatic separation medium, centrifuged after1640+10%autologous plasma absorb mononuclear cells cultured in37°C,5%CO2incubator for2hours, separated human peripheral blood mononeuclear cells(PBMC) from Human PeriPheral blood, then cultured the cells in1640medium containing10%for2hours.then collected and induced the non-adherent cells in1640medium containing10%Human plasma,add GM-CSF and IL-4. observed cells with light microscope, and use flow cytometry to measured their surface markers。Mixed mature DC with IL-13Ra2protein,tumor cell antigen and a tumor stem cell antigen, then cultured them for24hours to get DC vaccines. Isolated T cells from human peripheral blood mononeuclear cells (PBMC),then cultured T lymphocytes and DC in the RPMI1640containing10%Human plasma, compared anti-tumor effect with different DC vaccines.Statistical analysis:Results are expressed as mean±SD. The t test was used for comparisons of two groups,oneway ANOVA was used for Comparisons of multiple groups. Difference is considered significant at P<0.05
Results There was no expression of IL-13Ra2in all cases of normal brain tissues. However,33of the59glioma tissues showed positive expression of IL-13Ra2(55.9%), There were specially significant difference between them(P<0.01).duing the glioma group, the expression of IL-13Ra2was significantly different between low grade glioma (I-II grade) and high grade glioma (Ⅲ桰Vgrade)(P<0.01)。 U251cell line also showed positive expression of IL-13Ra2too. We successfully achieved enough U251cells and glioma cells by culturing them in serum containing medium, the cells seen under the microscope have shapes as spindle style, star style and polygonal style. The cell have translucent endochytema, and grown in the monolayer and adherence way on the bottle. It grows rapidly, passage in every week. After4th generation,we found the polygonal shape became the most shape in the cells. Eulturing glioma cells in serum-free medium we found BTSC grown as The cloning balls of cells,4th generation protuberance more visible, accounted for most of the polygonal shape. Serum-free stem cell cultured culture medium more than1week grown in suspension, formed the the suspensions tumor clone ball, the refractive better, flow cytometry cell markers CD133positive for expression rates of the different patients. flow cytometry found many CD133+cells in the brain tumor stem cells, Expression rate of low grade gliomas became higher with culture time increased, when cells was inoculated into serum containing medium, many cells move out of clone ball,The characterization of them was same as primary glioma cells。DCs could be isolated from PBMC by GM-CSF and IL-4. under the microscope we can seen the typical dendritic structure on DC's membrane surface.Flow cytometry detected the PBMCs surface marker, CD14, have low expression on DCs,while the expression rates of DCS characteristic surface markers higher than before,(CD8387.1%, CDla81%, CD8684.2%、 HLA-DR83%、CD8078%), we get large number of purity mature DC from PBMC.The Vitro Cells Cytotoxic experiment results show that, Compared with pure DC group, DC vaccines loaded by IL-13Ra2, glioma cell antigen and brain tumor stem cell antigen could significantly stimulated the proliferation of T lymphocytes, and abolish glioma cells Effectively. Compared to other groups, DC vaccines loaded by brain tumor stem cell antigen have the strongest effect to activated CTL (P<0.05), while IL-13Ra2group and tumor cell antigen group have no significant difference in the effect (P>0.05). IL-13Ra2group have weakly effect to induced CTL for glioma stem cell, when compare it with the pure DC group,we got no statistical significance from statistical analysis (P>0.05). The stimulation index of used brain tumor stem cell antigen was higher than others in the same stimulator to responder ratio, and when the ratio was1:10,the stimulation index was the highest one.
Conclusion:IL-13Ra2protein do not express in normal brain tissue,but widely expressed in glioma and U251cell line.The Expression of IL-13Ra2is associated with the malignant degrees of glioma, so it is an ideal immunotherapy target; We can get enough brain tumor stem cells in the way of culture Glioma cell and U251cell line in serum-free medium; Sour ce of of human peripheral blood mononuclear cells from in vitro GM-CSF and IL-4induce--d cultured available under plenty of mature DC dendritic cells provide a source of cells for tumor immunotherapy in DC vaccines. Compared to IL-13Ra2group and glioma cell antig-en, DC vaccines loaded by brain tumor stem cell antigen have the strongest effect to active-ted CTL which directed against glioma cell and brain tumor stem cell, besides, DC va-ccine loaded by IL-13Ra2and glioma cell antigen also have obviously effect to kill glio-mas cells,so they also have has potential value in clinical therapy.
引文
[1]王忠诚.神经外科学[M].武汉：湖北科学技术出版社,2005.497-586.
[2]Reto S, Gokhan Y,Silvia M. Neural stem cells,tumour stem cells and brain tumours: Dangerous relationships?[J].Biochimica et Biophysica Acta,2007,1776(3):125-137.
[3]Mao X G, Zhang X, Zhen H N. Progress on Potential strategies to target brain tumor stem cells[J].Cell Mol Neurobiol,2009529(2):141-155
[4]窦长武；树突状细胞介导的抗脑胶质瘤的免疫治疗进展 内蒙古医学院学报,Acta Academiae Medicinae Neimongol,2007,29 (6)452-455
[5]俞建波,冯志仙,詹仁雅,童鹰,马跃辉,潘剑威 RNA 致敏树突状细胞治疗胶质瘤的实验研究浙江大学学报(医学版),Journal of Zhejiang University(Medical Sciences),2007,36(6)92-97
[6]Bullard D E, Gillespie G Y, Mahaley M S, et al. Immunobiology of human gliomas[J].Semin Oncol,1986,13(1):94-109.
[7]Becher B, Prat A, Antel JP. Brain-immune connection:immuno-regulatory Properties of CNS resident cells. GLIA 2000 15;29(4):293-304
[8]Parajuli P, MathuPala S, Mittal S, et al. Dendritic cell-based active specific Immunotherapy for malignant glioma [J]. Expert Opin Biol Ther,2007,7(4):439-448.
[9]Gu J H, Li G Dendritic cell-based immunotherapy for malignant glioma [J].Neurosci Bull,2008,24(1):39-44.
[10]Tsugawa T, Kuwashima N,Sato H,et al. Sequential delivery of interferon-a gene and DCs to intracranial gliomas promotes an effective antitumor response[J].Gene Ther,2004,11:1551-1518.
[11]Steinman R M. The dendritic cell system and its role in immunogenicity[J].Annu Rev Immunol,1991,9:271-296
[12]Furumoto K, Mori A, Yamasaki S,et al. Interleukin-12-gene transduetion makes DCs from tumor-bearing mice an effective inducer of tumor-specific immunity in a Peritoneal dissemination model.Immounol Let,2002,83(1):13-20
[13]Held F J, Lutjohann B, Ungerfroren H, et al. Interaction of transforming growth factor-beta TGF-beta and epidermal growth factor EGF in human glioma cells[J]. J Neurooncol,2003; 632:117
[14]Yamanaka R, Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase IPII tria [J]. Clin CancerRes,2005; 11 (11):4160-4167
[15]Heimberger AB,Abou-Ghazal M, Reina-Ortiz C, et al. Incidence and Prognostic Impact of Fox P3+regulatory T cells in human gliomas.Clin Cancer Res 2008 15;14(16):5166-72
[16]刘福生,王忠诚,历俊华等。肿瘤抗原致敏树突状细胞瘤苗治疗颅内胶质瘤的实验研究。中华神经外科杂志,2002,18：91-95
[17]Yoshida S, Morii K, Watanabe M, et al. The generation of anti-tumoral cells using dentritic cells from the peripheral blood of patients with malignant brain tumors.Cancer Immunol Immunother.2001,50(6):321-327
[18]Yu JS, Wheeler CJ, Zeltzer PM, et al. Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res.2001,61(3):842-847
[19]Ardavin C, Amigorenas, Reis E S C. Dendritic cells:immunobiology and cancer immunotherany [J].Immunity,2004,20(1):17-23.
[20]牛洪泉,雷霆,王煜等。增强型绿色荧光蛋白基因对树突状细胞功能的研究[J].中华实验外科杂志,2006；23(5)：548-550
[21]牛洪泉,雷霆,王煜等。树突状细胞瘤内注射和化疗联合治疗脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(3)：184-186
[22]牛洪泉,雷霆,柳再明等。树突状细胞和小胶质细胞在脑胶质瘤自体免疫治疗中的作用机制[J].华中科技大学学报·医学版,2005； 34(6)：655-658
[23]牛洪泉,舒凯,董芳永等。树突状细胞治疗脑胶质细胞瘤实验研究[J].中华神经外科疾病研究杂志,2005；4(4)：309-312
[24]雷霆,牛洪泉,董芳永等。脑胶质瘤患者自体免疫治疗前后T细胞亚群的变化[J].肿瘤防治研究,2005；32(12)：782-784
[25]牛洪泉,王煜,董芳永等。大鼠树突状细胞疫苗治疗脑胶质瘤的实验研究[J]中国临床神经外科杂志,2005； 10(2)：115-117
[26]牛洪泉,董芳永,董震等。树突状细胞和脑胶质瘤自体免疫治疗的实验和临床研究[J].中华神经外科杂志,2004；20(2)：131-133
[27]张韬,牛洪泉,雷霆等。对负载人胶质瘤冻融抗原的树突状细胞功能的实验研究[J].中国临床神经外科杂志,2003；8(4)：242-244
[28]董震,牛洪泉,雷霆等。自体树突状细胞肿瘤疫苗治疗脑胶质瘤的临床初步观察[J].中国临床神经外科杂志,2003；8(4)：249-251
[29]董芳永,牛洪泉,董震等。脑胶质瘤特异性肿瘤疫苗的制备[J].中国临床神经外科杂志,2003；8(4)：252-254
[30]薛德麟,雷霆,董震等。脑胶质瘤自体疫苗的临床实验研究[J].中华实验外科杂志,2003；20(2)：139-140
[31]孟庆海,薄勇力,李洛等。树突状细胞疫苗治疗大鼠脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：55-58
[32]Ludewig B, Ochsenbein AF, Odermatt B, et al. Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease.J Exp Med,2000,191(5):795-804.
[33]Donaldson D D, Whitters M J, Fitz L J, etal. The murine IL-13 receptor alpha 2: molecular cloning, characterization, and compareson withmurine IL-13 receptor alpha 1 [J]. J Immuno,l 1998,161(5):2317-2324.
[34]Rahaman S O, Sharma P, Harbor P C, et a.l IL-13Ra2, a decoy receptor for IL-13 acts as an inhibitor or IL-4 dependent signal transduction in glioblastoma cells. CancerResearch,2002,62(4):1103-1109
[35]Joshi BH, Plautz GE, Puri RK. Interleukin-13 receptor alpha chain:a novel tumor-associated transmembrane protein in primary explants of human malignant gliomas [J]. CancerRes,2000,60(5):1168-1172.
[36]Shimato S, Natsume A, Wakabayashi T, et al. Identification of a human leukocyte antigen-A24-restricted T-cell epitope derived from interleukin-13 receptor alpha2 chain, a glioma-associated antigen[J]. J Neurosurg,2008,109(1):117-122.
[37]DebinskiW, Gibo D M, Puri R K. Novel way to increase targeting specificity to a human glioblastoma-associated receptor for interleukin 13. Int JCancer,1998,76(4): 547-551
[38]Husain SR, Joshi BH, Puri R K. Interleukin-13 receptor as a unique target foranti-glioblastoma therapy. IntJ Cancer,2001,92(2):168-175
[39]Lapidot T,Sirard C,Vormor J.et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice [J]. Nature,1994,367(6464):645-648
[40]Dick J E. Acute myeloid leukemia stem cells [J]. Ann N Y A cad Sci,2005.1044:1-5
[41]Ignatova TN, Kukekov VG, Laywell ED,et al Human cortical glial tumors contain neural stem-like cells expressing astroglial and neural markers in vitro.Glia 2002;39:193-206
[42]Nakagawa, M. Koyanagi, M.Tanabe, K. et al. Generation of induced PluriPotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol.2008;26:101-106.
[43]Singh S K, Hawkins C, Clarke I D, et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396-401
[44]Singh S K, Clarke I D, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors[J]. Cancer Res,2003,63(18):5821-5828
[45]Viehehatom P, Wongkajornsilp A, Petvises S, et al. Dendritic cells Pulsed with total tumor RNA for activation NK-like T cells against glioblastoma multiforme[J].J Neurooncol,2005,75(2):111-118.
[46]Garcia-Hernandez M L, Gray A, Hubby B, et al. Prostate stem cell antigen Vaccination induces a long-term Protective immunune response against Prostate Cancer in the absence Of autoimmunity[J]. Cancer Res,2008,68(3):861-869.
[47]Ahmed et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clin Cancer Res.2010 Jan 15;16(2):474-85. Epub 2010 Jan 12.
[48]Pellegatta S, Poliani PL, Como D,et al. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.Cancer Res,2006,66(21):10247-10252.
[1]陈忠平周旺宁我国胶质瘤诊断治疗现状和努力方向中国肿瘤,Bulletin of Chinese Cancer,2005,14 (2) 78-81
[2]胡永生,张庆林,田志刚,等.TH1/TH2类细胞因子在脑胶质瘤中的不均衡表达及其意义[J].中国医学科学院学报,2001,23(6)：594-598.
[3]Oshima Y, Joshi BH, Puri RK. Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution[J]. J Biol Chem,2000,275(19):14375-14380.
[4]Terabe M.et al. Role of IL-13 in regulation of anti-tumor immunity and tumor geowth [J]. Cancer Immunol Immunother,2004,53(2):79-85.
[5]Kawakami K, Taguchi J. Murata T, et al. The interleukin-13 receptor alpha 2 chain: an essential component for binding and internalization but not for interleukin a-13-induced signal transduction through the STAT6 pathway[J]. Blood,2001,97(9):2673-2679.
[6] Rahaman SO, Sharma P, Harbor PC, et al. IL-13R(alpha)2, a decoy receptor for IL-13 acts as an inhibitor of IL-4-dependent signal transduction in glioblastoma cells[J]. Cancer Res,2002,62(4):1103-1109.
[7]Joshi BH, Plautz GE, Puri RK. Interleukin-13 receptor alpha chain:a novel tumor-associated transmembrane protein in primary explants of human malignant gliomas[J]. Cancer Res,2000,60(5):1168-1172
[8]Hao C, et al.Cytokine and cytokine receptor mRNA expression in human glioblas-tomas:evidence of Thl, Th2 and Th3 cytokine dysregulation. ActaNeuropathol(Berl) 2002,103(2):171-8
[9]Debinski W, Slagle B, Gibo DM, et al. Expression of a restrictive receptor for interleukin 13 is associated with glial transformation[J]. J Neurooncol,2000,48(2):103-111.
[10]Liu H,Jacobs B S,Liu J,et al. Interleukin-13 sensitivity and receptor phenotypes of human glial cell lines:non-neoplastic glia and law-grade astrocytoma differ from malignant glioma.Cancer Immunol Immunother,2000,49:319-324
[11]Kawakami M, Kawakami K,Takahashi S,et al. Analysis of interleukin-13 Receptor alpha2 Expression in human pediatric brain tumors.Cancer,2004,101:1036-1042.
[12]Saiali S,Avril T,Collet B,et al.Expression of nine tumour antigens in a series of human glioblastoma multiforme:Interest of EGFRVIII,IL-13 Ralpha2,gp100,and TRP-2 for inmmunotherapy.J Neurooncol,2007,81:139-148
[1]王忠诚主编.王忠诚神经外科学[M].武汉：湖北科学技术出版社,2005
[2]Mao X G, Zhang X, Zhen H N. Progress on Potential strategies to target brain tumor stem cells[J].Cell Mol Neurobiol,2009,29(2):141-155
[3]Hegedus B, Dasgupta B, Shin J E, et al. Neurofibromatosis-1 regulates neuronal and glial cell differentiation from Neuroglial Progenitors in vivo by both cAMP and Ras-dependent mechanisms. Cell Stem Cell 007:1:443-457
[4]Singec 1, Knoth R, Meyer RP,et al.Defining the actual sensitivity and specificity Of the neurosphere assay in stem cell biology.Nat Methods 2006;3:801-6
[5]Ignatova TN, Kukekov VG, Laywell ED,et al Human cortical glial tumors contain neural stem-like cells expressing astroglial and neural markers in vitro.Glia 2002;39:193-206
[6]Hambardzumyan D,Squatrito M, Holland E C.Radiation resistance and Stem-like cells in brain tumors[J].Cancer Cell,2006,10(6):454-456.
[7]Pellegatta S, Poliani PL, Como D,et al. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.Cancer Res,2006,66(21):10247-10252.
[8]Bernard DJ, Courjal F, Maurizis JC, et al. Effect of epidermal growth factor in HLA class I and class II transcription and protein pression in human breast adenoeareinoma cell lines. Br J Cancer 1992,66:88-92
[9]康德智,苏东辉等.高表达Hsp70鼠脑胶质瘤细胞瘤苗的体外抗瘤作用[J].中华神经医学杂志,2003.2(1)：52-56.
[10]Heimberger AB,Sampson JH.The PEPvⅢ-KLH(CDX-110) vaccine in Gliob-lastoma multiforme Patients. Expert OPin Biol Ther.2009:9(8):1087-1098
[11]Roszman TL, Brooks WH,Elliott LH.Immunobiology of Primary in traeranial tumors.VI.Suppressor cell function and lectin-binding lymphocyte Subpopulations in patients with cerebral tumors. Cancer 1982 Oct 1:50(7):1273-9.
[12]Jordan C T, Guzman M L, Noble M. Cancer stem cells[J].N Engl J Med,2006,355 (12) :1253-1261.
[13]Xie Z.Brain tumor stem cells [J]. Neurochem Res,2009,34(12):2055
[14]Bonnet D,Diek JE.Human acute myeloid leukemia is organized as A hierarehy that originates from a primitive hematopoietic cell[J].Nature Med,1997,3(7):730-737.
[15]Al-Hajj M, Wicha MS, Benito HA, et al. Prospcetive Identification of tumorigenic breast cancer cells[J].Proc Natl Acad Sci USA,2003,100(7):3983-3988.
[16]Singh S K, Hawkins C, Clarke I D, et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396-401
[17]Veseovi AL, Galli R, Reynolde BA, et al. Brain tumor stem cell[J]. Nature,2006,6(17):425-436.
[18]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133+cancer stem cells in glioblastoma [J]. Mol Cancer,2006,5:67.
[19]Bao S,Wu Q,Melendon R E,et al.Glioma stem cells promote radioresistance by Preferential activation of the DNA damageresponse[J].Nature,2006,444(7120):756-760.
[20]Brewer GJ Isolation and culture of adult rat hippocampal neurons [J]. Neurosci, Methods,1997,71(2):143-155
[21]Groszer M, Erickson R, Scripture-Adams DD, et al. Negative regulation of neural stem Progenitor cell Proliferation by the Pten tumor suppressor gene in vivo[J]. Science,2001 294(5549):2186-189.
[22]Li C, Heidt DG, Daletha P, Burant CF, et al Identification of Pancreatic caneer Stemcells.Cancer Res 2007;67:1030-1037
[23]Singh S K, Hawkins C, Clarke I D, et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396-401
[24]Wan F, Zhang S, xie R, et al. The utility and limitations of Neurosphere assay, CD133 immunophenotyping and side population assay in glioma stem cell Research[J].Brain pathol,2010,20(5):877-889.
[25]CamPos B,Herold-MendeC C.Insight into the complex regulation of CD133 in glioma [J].Int J Cancer,2011,128(3):501-510.
[26]张全斌,黄强,兰青.脑肿瘤干细胞的热点问题及研究进展[J].中华医学杂志,2005(26)：1868-1870.
[27]Figdor CG, de Vries IJ, Lesterhuis WJ, et al. Dendritic cell immunotherapy:mapping the way.Nat Med 2004; 10:475-80.
[1]孟凡东,隋承光,王晓华等.体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究[J].中国年学杂志,2006;26(6)：818-820
[2]Stupp R, Hegi M E. Targeting brain-tumor stem cells [J]. Nat Biotechnol, 2007,25(2):193-194
[3]刘福生,金贵善,历俊华等.树突状细胞与反义TGF-β1基因修饰的胶质瘤细胞融合瘤苗治疗脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：47-50
[4]俞建波,冯志仙,詹仁雅,童鹰,马跃辉,潘剑威 RNA 致敏树突状细胞治疗胶质瘤的实验研究浙江大学学报(医学版),Journal of Zhejiang University(Medical Sciences),2007,36(6)92-97
[5]Steinman RM, Cohn ZA. Identification of a novel cell type in Peripheral lymphoid organs of mice. MorPhology, quantitation, tisSued distribution [J].J Exp Med,1973,137(5):1142-1162.
[6]Walker PR,Calzascia T,Schnuriger V, et al. The brain parenchyma is permissive for full antitumor CTL effector function,even in the absence of CD4 T cells. [J].Immunol.2000;165:3128-3135
[7]Brossart P,Wirths S,Brugger W,et al. Dendritic cells in cancer vaccines[J] ExP Hematol,2001,29(11):1247-1255.
[8]Dendritic cells pulsed with Total tumor RNA for activation NK-like T cells against glioblastoma multiforme[J].J Neurooncol,2005,75(2):111-118
[9]Berney C, Herren S, Power CA, et al. A member of the dendritic cell family that enters B cell follicles and stimulates Primary antibody responses identified by a mannose receptor fusion Protein [J] J ExP Med,1999,190(6):851-860.
[10]Kiertscher SM, Luo J, Dubinett SM, et al.Tumors promote altered maturation and early apoprosis of monocyte derived dendritic cells[J].J Immununol,2001,164(7):1269-1277.
[11]Steinman RM, Turley S, Mellmanl,et al.The induction of tolerance by dendritic cells tha thave captured apoptotic cells. J.Exp.Med.2000; 191:411-416
[12]Hagihara M, Li C, Gansuvd B, et al. Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma [J].Immunol Methods,2001,253(1-2):45-55.
[13]Sehendel DJ. Dendritic cell vaccine strategies for renal cell careinoma[J).Expert opin Biol Ther,2007,7(2):221-232.
[14]Koido S, Hara E, Homma S, et al. Dendritic/tumor fusion cell-based vaccination against cancer[J].Arch Immunol Ther Exp(Warsz),2007,55(5):281-287.
[15]Ardavin C,Amigorena S, Reis E S C. Dendritic cells:immunobiology and cancer immunotherany[J].Immunity,2004,20(1):17-23.
[16]Holland EC, Celestino J, Dai C,et al. Combined activation of Ras and Akt in neural progenitors Induces glioblastoma formation in mice[J]. Nat.Genet.2000,25(l):55-57
[17]冯志华,王全楚主编.新概念疫苗[M].北京：人民卫生出版社,2004.
[18]Banchereau J, Briere F, Caux C, et al.Immunobiology of dendritic cells[J].Annu Rev 1 mmunol,2000,18:767-811.
[19]Liau LM,Prins RM,Kiertscher SM, et al.Dendritic cell vaccination in glioblastoma patients induces systemic and intraecranial T-cell responses modulated by the local central nervous system tumor microenvironment. Clin Cancer Res 2005:11(15):5515-25.
[20]王东海,李新钢,曲迅,等.树突状细胞融合瘤苗抗胶质瘤的实验研究.中华神经外科杂志,2006,22：699-701.
[21]Yamanaka R, Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase IPII tria [J]. Clin CancerRes,2005; 11 (11):4160-4167
[22]Ashley D M, Faiola B, Nair S, et al. Bone marrow-Generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors[J]J Exp Med,1997,186(7):1177-1182
[23]Heimberger AB,Abou-Ghazal M, Reina-Ortiz C, et al. Incidence and Prognostic Impact of Fox P3+regulatory T cells in human gliomas.Clin Cancer Res 2008 15;14(16):5166-72
[24]Pellegatta S, Poliani PL, Como D,et al. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.Cancer Res,2006,66(21):10247-10252.
[1]Jeremias I, Steiner HH, BennerA, et al. Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells [J]. Acta Neurochir,2004; 146 (7):721-729
[2]姜政,江玉泉,李新钢,等.人脑胶质瘤细胞原代培养实验模型的构建[J].中华肿瘤防治杂志,2006；13(10)：744-747
[3]陈忠平周旺宁我国胶质瘤诊断治疗现状和努力方向 中国肿瘤,Bulletin of Chinese Cancer,2005,14, (2) 78-81
[4]窦长武；树突状细胞介导的抗脑胶质瘤的免疫治疗进展 内蒙古医学院学报,Acta Academiae Medicinae Neimongol,2007,29 (6) 452-455
[5]Lendah U, Zimmerman LB, Mckay RD. CNS stem cell express a new class of intermediate filament protein[J]. Cell,1990,60(4):585-595
[6]Recht L, Jang T, Savarese T, et al. Neural stem cells and neuIo-on-eulogy:quo vadis[J]. J Cell Biochem,2003,88(1):11-19.
[7]于春泳,魏学忠,宋振全.成熟大鼠脑损伤后神经干细胞的表达与增殖[J].中国临床康复,2002,6(2)：1744-1745.
[8]Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell [J]. Nat Med,1997,3(7):730-737.
[9]Matsui W, Huff CA, Wang Q, et al. Characterization of clonogenic multiple myeloma cells[J]. Blood,2004,103(6):2332-2336.
[10]AI-Hajj M, Wicha MS, Benito-Hernandez A, et ol. Prospective identification of tumorigenic breast cancer cells[J]. Proc Natl Acad Sci USA.2003,100(7): 3547-3549.
[11]Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells[J]. Nature,2001,404(6859):105-111.
[12]Ignatova TN, Kukekov VG, Laywell ED, et al. Human cOf tical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro [J]. Glia, 2002,393; 193-206.
[13]Singh SK, Clarke ID, Terasaki M, et ol. Identification of cancer stem cell in human brain tumors [J]. Cancer Res 2003; 63181:5821-5828.
[14]Hemmati HD, Nakano I, Lazareff JA, Cancerous stem cells can arise from pediatric brain tumors[-[J]. Proc Natl Acad Sci USA,2003,100(25):15178-15183.
[15]Yi L, Zhou ZH, Ping YF, et al. Isolation and characterization from primary human anaplastic oligoastroeytoma [J]. Modern Pathology,2007,20:1061-1068.
[16]Kondo T, Setoguchi T, Taga T. Persistence of a small sub-population of cancer stein-like ceils in the C6 glioma cell line[J]. Proct Natl Acad Sci USA,2004,101(3): 781-786.
[17]Ghods AJ, Irvin D, IAu G, et cd. Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells [J]. Stem Cells,2007, 25(7):1645-1653.
[18]Yu SC, Ping YF, Yi L, et al. Isolation and characterization of cancer stem cells from U87[J]. Cancer Letters,2008,265:124-134.
[19]STROJNIK T, ROSLAND G V, SAKARIASSEN P O, et a 1. Neural stem cell markers, nestin, and musashi proteins, in the progression of hum-an glioma: correlation of nestin with prognosis of patient s-rvival[J]. Surg Neurol,2007,68 (2): 133-144.
[20]SINGH S CLARKE ID, HIDE T, et al. Cancer stem cells in nervous system tumors [J]. Oncogene,2004,23(43):7267-7273
[21]BERGER F, GAY E, PELLETIER L, et al. Development of gliomas:potential role of asymmetrical cell division of neural stem cells [J]. lancet Oncol,2004,5(8):511-5 14.
[22]黄强,董军,朱玉德,等.人脑胶质瘤组织中分离与培养肿瘤干细胞[J].中华肿瘤杂志,2006,28(5)：331-333.
[23]Raso A, Negri F, Gregorio A, et al. Successful isolation and long-term establishment of a cell line with stem cell, like features from all anaplastic medulloblastoma[J J. Neuropathol Appl Neurobiol,2008,34(3):306-315.
[24]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133 cancer stem cells in glioblastoma[J]. Mol Cancer, 2006,5:67.
[25]周志华,易良,卞修武,等.人恶性胶质瘤细胞株U251三种克隆的分化特性和胶质瘤干细胞的初步鉴定[J].中华神经外科杂志,2008,24(9)：658-662.
[26]Yuan XP, Curlin J, Xiong YZ, et al. Isolation of cancer stem cells from adult glioblastoma multiform [J]. Oncogene,
[27]季晓燕,黄强,董军等,脑肿瘤干细胞体外分化的形态标志物及细胞增殖动力学特征[J].中华医学杂志,2006,86(23)：1604-1609.
[28]Barnett S C, Robertson L, Graham D, Allan D, and Rampling R Oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells transform ed with c-myc an d H-ras form high-grade glioma after stereotactic injection into the rat [J]. Carcinogenesis,1998,19(9):1529-1537.
[29]Barnett S C, Robertson L, Graham D, Allan D, and Rampling R Oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells transform ed with c-myc an d H-ras form high-grade glioma after stereotactic injecfion into the rat brain[J] Carcinogenesis,1998,19(9):1529-1537.
[30]Beier D, Hau P, Proescholdt M. CD133 and CD133-glioblastoma. derived cancer stem ceUs show differential growth characteristics and molecular profiles[J]. Cancer Res,2007,67(9):4010-4015.
[31]Wechsler-Reya RJ. Analysis of gene expression in the normal and malignant cerebellum [J]. Recent Prog Horm Res,2003; 58:227-248.
[32]Lubensky IA, Vortmeyer AO, Kim S, et ol. Identification Of tumor precursor cells in the brains of primates with radia. Tion-induced de novo glioblastoma multiforme[J]. Cell Cycle,2006; 5(4):452-456.
[33]Aboody KS, Brown A, Rainov NG, et ol. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas[J]. Proc Natl Acad Sci US-A.2000; 97(23):12846-12851.
[34]Hanahan D, Weinbery RA. The hallmarks of cancer J.Cell,20000,100;57-70
[35]李茗初,邓永文,伍军等.髓母细胞瘤中脑肿瘤干细胞的分离培养及鉴定[J].癌症,2006；25(2)：241-246.
[36]Bschoo RM,Maher EA, Ligon KL, et al Epidermal groeth factor receptor and Ink4a/Aft [J] Cancer cell 2002,1,(3):269-277
[37]Holland EC. Gliomagenesis:genetic alterations and mouse models[J]. Nat Rev Genet, 2001; 2(1):120-129.
[38]Nagakawa M,Kpyangagi M.,et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts J Nat Biotechnol 2008,26(1):101-106
[39]Hope KJ,Jin LQ, Dick JE, et al. Acute myeloid Leukemia originate from a hierarchy of leukemic stem cell classess that differ in self-renewal capccity J Nature Imunol,2004,5:738-743
[40]Joo KM, Kim SY, Jin X, et al. Clinical and biological implications of CDI33-positive and CDI33-negative cells in glinblastomas[J]. Lab Invest,2008,88(8):808-815.
[41]Zheng X, Shen G, Yang X, et al. Most C6 cells are cancer stem cells:evidence from clonal and population analyses[J]. Cancer Res,2007,67(8):3691-3697.
[42]Wang J, Sakariassen PQ, Tsinkalovsky O, et al CD 133 negative glioma cells from tumors in nude rats and give rise to CD133 positive cells[J]. Int J Cancer,2008, 122(4):761-768.
[43]陈骅,黄强.胶质瘤干细胞研究的新观点[J].中国神经肿瘤杂志,2008,1(6)：1-4.
[44]Shervington A, Lu C. Expression of multidrug resistance genes in normal and cancer stem cells[J]. Cancer Invest,2008,26(5):535542
[45]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133 cancer stem cells in glioblastoma[J]. Mol Cancer,2006,5:67.
[46]Lu C, Sheiwington A. Chemoresistance in gliomas[J]. Mol Cell Biochem, 2008.312(1-2):71-80
[47]Bi CL, Fang JS, Chen FH, et al. Chemoresistance of CD133(+) tumOr stern cells from human brain glioma[J]. Zhong Nan Da Xue Xue Bao Yi Xue Ban,2007,32(4): 568-573.
[48]Beier D, Ri-hrl S, Pillai DR, et al. Temozolomide preferentially depletes cancer stem cells in glioblastoma[J]. Cancer Res,2008,68(14):5706-5715.
[49]Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response [J]. Nature,2006,444(7120): 756-760.
[50]Blazek ER. Foutch JL. Maki G. Daoy medulloblastoma cells that express CD133 are radioresistant relative to CD133-cells, and the CD133+sector is enlarged by hypoxia[J]. Int J Radiat Oncol Biol Phys,2007,67(1):1-5.
[51]Hambardzumyan D, Becher OJ, Rosenblum MK, et al. PI3 K pathway regulates survival of cancer stenl cells residing in the perivascular niche following radiation in medulloblastoma in vivo[J]. Genes Dev,2008,22(4):436-448.
[51]KleinRS,LinE,ZhangB,etal.Neuronal CXCL10 directs CD8+T-cell recruitment and control of West Nile virus encephalitis [J]. JVirol,2005,79:11457-11466.
[52]SeoDR, KimKY, LeeYB.Interleukin-10 expression in lipopolysaccharide-activated microglia is mediated by extracellular ATP in an autocrine fashion [J] Neuroreport,2004,15:1157-1161.
[53]Tsugawa T,KuwashimaN,SatoH,etal.Sequential de-livery of interferon-a gene and DCs to intracranial gliomas promotes an effective antitumor response[J].Gene Ther,2004,11:1551-1518.
[54]刘福生,金贵善,历俊华,等.树突状细胞与反义TGF-β1基因修饰的胶质瘤细胞融合瘤苗治疗脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：47-50
[55]俞建波,冯志仙,詹仁雅,童鹰,马跃辉,潘剑威RNA致敏树突状细胞治疗胶质 瘤的实验研究浙江大学学报(医学版),Journal of Zhejiang University(Medical Sciences),2007,36(6)92-97
[56]YANG B Y, WU CY。The effect of vaccine adjuvants such as IL.12and CpG to CD4+ Tcells induced specific immune response in vivo[J].Chin J Immunol,2005,11: 803-807.
[57]孟凡东,隋承光,王晓华等.体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究[J].中国年学杂志,2006；26(6)：818-820
[58]Held FJ,Lutjohann B,Ungerfroren H, et al. Interaction of transforming growth factor-beta TGF-beta and epidermal growth factor EGF in human glioma cells[J]. J Neurooncol,2003; 632:117
[59]Yamanaka R,Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase IPII tria [J]. Clin CancerRes,2005; 11 (11):4160-4167
[60]宫安静,孟庆海,朱湘华,等.树突状细胞诱导的CTLs与CIK及LAK细胞对神经胶质瘤杀伤作用比较[J].青岛大学医学院学报,2007；43(2)：159-161
[61]于音,赵刚多基因联合在胶质瘤治疗中的应用进展山东医药,Shandong Medical Journal,2007年33期
[62]Parajuli P, Mathupala S, Sloan AE. Systemic comparison of dendritic cell-based immunotherapeutic strategies for malignant gliomas:in vitro induction of cytolytic and natural killer-like T cells. Neurosurgery,2004,55(5):1194-1204
[63]Saito R, Mizuno M, Nakahara N, et al. Vaccination with tumor cell lysate-pulsed dendritic cells augments the effect of IFN-beta gene therapy for malignant glioma in an experimental mouse intracranial glioma. Int J Cancer,2004,111(5):777-82
[64]孟庆海,薄勇力,李洛等.树突状细胞疫苗治疗大鼠脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：55-58
[19]Yu JS,Wheeler CJ, Zeltzer PM. et al. Vaccination of malignant glioma patients with peptide-pulsed dendritcells elicits systemic cytotoxicity and intracranial T-cell infiltration[J]. CancerRes,2001;613:842-847
[65]赵丽波,李才,周维国树突状细胞负载的肿瘤抗原肽疫苗抗脑胶质瘤作用的实验研究中国实验诊断学,[J]. Chinese Journal of Laboratory Diagnosis, 2009.01(1):112-114
[66]Akasaki Y, Kikuchi T, Homma S, et al. Antitumor effect of im-munizations with fusion of dendritic and glioma cells in a mouse brian tumor model. J Immunother,2001, 24(2):106-113.
[67]Kjaergaard J,WangLX, Kuriyama H, et al. Active immunotherapy for advanced intracranial murine tumors by using dendritic cell-tumor cell fusion vaccines [J]. J Neurosurg,2005;103 (1):156-164
[68]RyuyaYamanaka Cell-and peptide-based immunotherapeutic approaches for glioma
[69]Wilm Mae, DC vaccination with anti-CD25 treatment leads to long-term immunity against experimental glioma, Neuro Oncol 2009, DOI:10.1215/15228517-2009-004
[70]张晓实树突细胞疫苗治疗脑胶质瘤的研究进展中国神经肿瘤杂志,Chinese Journal of Neuro-Oncology,2007; 5 (3) 149-151
[71]Ludewig B, Ochsenbein AF, Odermatt B, et al. Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease. [J]Exp Med,2000,191 (5):795-804.
[2]Reto S, Gokhan Y,Silvia M. Neural stem cells,tumour stem cells and brain tumours: Dangerous relationships?[J].Biochimica et Biophysica Acta,2007,1776(3):125-137.
[3]Mao X G, Zhang X, Zhen H N. Progress on Potential strategies to target brain tumor stem cells[J].Cell Mol Neurobiol,2009529(2):141-155
[4]窦长武；树突状细胞介导的抗脑胶质瘤的免疫治疗进展 内蒙古医学院学报,Acta Academiae Medicinae Neimongol,2007,29 (6)452-455
[5]俞建波,冯志仙,詹仁雅,童鹰,马跃辉,潘剑威 RNA 致敏树突状细胞治疗胶质瘤的实验研究浙江大学学报(医学版),Journal of Zhejiang University(Medical Sciences),2007,36(6)92-97
[6]Bullard D E, Gillespie G Y, Mahaley M S, et al. Immunobiology of human gliomas[J].Semin Oncol,1986,13(1):94-109.
[7]Becher B, Prat A, Antel JP. Brain-immune connection:immuno-regulatory Properties of CNS resident cells. GLIA 2000 15;29(4):293-304
[8]Parajuli P, MathuPala S, Mittal S, et al. Dendritic cell-based active specific Immunotherapy for malignant glioma [J]. Expert Opin Biol Ther,2007,7(4):439-448.
[9]Gu J H, Li G Dendritic cell-based immunotherapy for malignant glioma [J].Neurosci Bull,2008,24(1):39-44.
[10]Tsugawa T, Kuwashima N,Sato H,et al. Sequential delivery of interferon-a gene and DCs to intracranial gliomas promotes an effective antitumor response[J].Gene Ther,2004,11:1551-1518.
[11]Steinman R M. The dendritic cell system and its role in immunogenicity[J].Annu Rev Immunol,1991,9:271-296
[12]Furumoto K, Mori A, Yamasaki S,et al. Interleukin-12-gene transduetion makes DCs from tumor-bearing mice an effective inducer of tumor-specific immunity in a Peritoneal dissemination model.Immounol Let,2002,83(1):13-20
[13]Held F J, Lutjohann B, Ungerfroren H, et al. Interaction of transforming growth factor-beta TGF-beta and epidermal growth factor EGF in human glioma cells[J]. J Neurooncol,2003; 632:117
[14]Yamanaka R, Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase IPII tria [J]. Clin CancerRes,2005; 11 (11):4160-4167
[15]Heimberger AB,Abou-Ghazal M, Reina-Ortiz C, et al. Incidence and Prognostic Impact of Fox P3+regulatory T cells in human gliomas.Clin Cancer Res 2008 15;14(16):5166-72
[16]刘福生,王忠诚,历俊华等。肿瘤抗原致敏树突状细胞瘤苗治疗颅内胶质瘤的实验研究。中华神经外科杂志,2002,18：91-95
[17]Yoshida S, Morii K, Watanabe M, et al. The generation of anti-tumoral cells using dentritic cells from the peripheral blood of patients with malignant brain tumors.Cancer Immunol Immunother.2001,50(6):321-327
[18]Yu JS, Wheeler CJ, Zeltzer PM, et al. Vaccination of malignant glioma patients with peptide-pulsed dendritic cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res.2001,61(3):842-847
[19]Ardavin C, Amigorenas, Reis E S C. Dendritic cells:immunobiology and cancer immunotherany [J].Immunity,2004,20(1):17-23.
[20]牛洪泉,雷霆,王煜等。增强型绿色荧光蛋白基因对树突状细胞功能的研究[J].中华实验外科杂志,2006；23(5)：548-550
[21]牛洪泉,雷霆,王煜等。树突状细胞瘤内注射和化疗联合治疗脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(3)：184-186
[22]牛洪泉,雷霆,柳再明等。树突状细胞和小胶质细胞在脑胶质瘤自体免疫治疗中的作用机制[J].华中科技大学学报·医学版,2005； 34(6)：655-658
[23]牛洪泉,舒凯,董芳永等。树突状细胞治疗脑胶质细胞瘤实验研究[J].中华神经外科疾病研究杂志,2005；4(4)：309-312
[24]雷霆,牛洪泉,董芳永等。脑胶质瘤患者自体免疫治疗前后T细胞亚群的变化[J].肿瘤防治研究,2005；32(12)：782-784
[25]牛洪泉,王煜,董芳永等。大鼠树突状细胞疫苗治疗脑胶质瘤的实验研究[J]中国临床神经外科杂志,2005； 10(2)：115-117
[26]牛洪泉,董芳永,董震等。树突状细胞和脑胶质瘤自体免疫治疗的实验和临床研究[J].中华神经外科杂志,2004；20(2)：131-133
[27]张韬,牛洪泉,雷霆等。对负载人胶质瘤冻融抗原的树突状细胞功能的实验研究[J].中国临床神经外科杂志,2003；8(4)：242-244
[28]董震,牛洪泉,雷霆等。自体树突状细胞肿瘤疫苗治疗脑胶质瘤的临床初步观察[J].中国临床神经外科杂志,2003；8(4)：249-251
[29]董芳永,牛洪泉,董震等。脑胶质瘤特异性肿瘤疫苗的制备[J].中国临床神经外科杂志,2003；8(4)：252-254
[30]薛德麟,雷霆,董震等。脑胶质瘤自体疫苗的临床实验研究[J].中华实验外科杂志,2003；20(2)：139-140
[31]孟庆海,薄勇力,李洛等。树突状细胞疫苗治疗大鼠脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：55-58
[32]Ludewig B, Ochsenbein AF, Odermatt B, et al. Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease.J Exp Med,2000,191(5):795-804.
[33]Donaldson D D, Whitters M J, Fitz L J, etal. The murine IL-13 receptor alpha 2: molecular cloning, characterization, and compareson withmurine IL-13 receptor alpha 1 [J]. J Immuno,l 1998,161(5):2317-2324.
[34]Rahaman S O, Sharma P, Harbor P C, et a.l IL-13Ra2, a decoy receptor for IL-13 acts as an inhibitor or IL-4 dependent signal transduction in glioblastoma cells. CancerResearch,2002,62(4):1103-1109
[35]Joshi BH, Plautz GE, Puri RK. Interleukin-13 receptor alpha chain:a novel tumor-associated transmembrane protein in primary explants of human malignant gliomas [J]. CancerRes,2000,60(5):1168-1172.
[36]Shimato S, Natsume A, Wakabayashi T, et al. Identification of a human leukocyte antigen-A24-restricted T-cell epitope derived from interleukin-13 receptor alpha2 chain, a glioma-associated antigen[J]. J Neurosurg,2008,109(1):117-122.
[37]DebinskiW, Gibo D M, Puri R K. Novel way to increase targeting specificity to a human glioblastoma-associated receptor for interleukin 13. Int JCancer,1998,76(4): 547-551
[38]Husain SR, Joshi BH, Puri R K. Interleukin-13 receptor as a unique target foranti-glioblastoma therapy. IntJ Cancer,2001,92(2):168-175
[39]Lapidot T,Sirard C,Vormor J.et al.A cell initiating human acute myeloid leukaemia after transplantation into SCID mice [J]. Nature,1994,367(6464):645-648
[40]Dick J E. Acute myeloid leukemia stem cells [J]. Ann N Y A cad Sci,2005.1044:1-5
[41]Ignatova TN, Kukekov VG, Laywell ED,et al Human cortical glial tumors contain neural stem-like cells expressing astroglial and neural markers in vitro.Glia 2002;39:193-206
[42]Nakagawa, M. Koyanagi, M.Tanabe, K. et al. Generation of induced PluriPotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol.2008;26:101-106.
[43]Singh S K, Hawkins C, Clarke I D, et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396-401
[44]Singh S K, Clarke I D, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors[J]. Cancer Res,2003,63(18):5821-5828
[45]Viehehatom P, Wongkajornsilp A, Petvises S, et al. Dendritic cells Pulsed with total tumor RNA for activation NK-like T cells against glioblastoma multiforme[J].J Neurooncol,2005,75(2):111-118.
[46]Garcia-Hernandez M L, Gray A, Hubby B, et al. Prostate stem cell antigen Vaccination induces a long-term Protective immunune response against Prostate Cancer in the absence Of autoimmunity[J]. Cancer Res,2008,68(3):861-869.
[47]Ahmed et al. HER2-specific T cells target primary glioblastoma stem cells and induce regression of autologous experimental tumors. Clin Cancer Res.2010 Jan 15;16(2):474-85. Epub 2010 Jan 12.
[48]Pellegatta S, Poliani PL, Como D,et al. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.Cancer Res,2006,66(21):10247-10252.
[1]陈忠平周旺宁我国胶质瘤诊断治疗现状和努力方向中国肿瘤,Bulletin of Chinese Cancer,2005,14 (2) 78-81
[2]胡永生,张庆林,田志刚,等.TH1/TH2类细胞因子在脑胶质瘤中的不均衡表达及其意义[J].中国医学科学院学报,2001,23(6)：594-598.
[3]Oshima Y, Joshi BH, Puri RK. Conversion of interleukin-13 into a high affinity agonist by a single amino acid substitution[J]. J Biol Chem,2000,275(19):14375-14380.
[4]Terabe M.et al. Role of IL-13 in regulation of anti-tumor immunity and tumor geowth [J]. Cancer Immunol Immunother,2004,53(2):79-85.
[5]Kawakami K, Taguchi J. Murata T, et al. The interleukin-13 receptor alpha 2 chain: an essential component for binding and internalization but not for interleukin a-13-induced signal transduction through the STAT6 pathway[J]. Blood,2001,97(9):2673-2679.
[6] Rahaman SO, Sharma P, Harbor PC, et al. IL-13R(alpha)2, a decoy receptor for IL-13 acts as an inhibitor of IL-4-dependent signal transduction in glioblastoma cells[J]. Cancer Res,2002,62(4):1103-1109.
[7]Joshi BH, Plautz GE, Puri RK. Interleukin-13 receptor alpha chain:a novel tumor-associated transmembrane protein in primary explants of human malignant gliomas[J]. Cancer Res,2000,60(5):1168-1172
[8]Hao C, et al.Cytokine and cytokine receptor mRNA expression in human glioblas-tomas:evidence of Thl, Th2 and Th3 cytokine dysregulation. ActaNeuropathol(Berl) 2002,103(2):171-8
[9]Debinski W, Slagle B, Gibo DM, et al. Expression of a restrictive receptor for interleukin 13 is associated with glial transformation[J]. J Neurooncol,2000,48(2):103-111.
[10]Liu H,Jacobs B S,Liu J,et al. Interleukin-13 sensitivity and receptor phenotypes of human glial cell lines:non-neoplastic glia and law-grade astrocytoma differ from malignant glioma.Cancer Immunol Immunother,2000,49:319-324
[11]Kawakami M, Kawakami K,Takahashi S,et al. Analysis of interleukin-13 Receptor alpha2 Expression in human pediatric brain tumors.Cancer,2004,101:1036-1042.
[12]Saiali S,Avril T,Collet B,et al.Expression of nine tumour antigens in a series of human glioblastoma multiforme:Interest of EGFRVIII,IL-13 Ralpha2,gp100,and TRP-2 for inmmunotherapy.J Neurooncol,2007,81:139-148
[1]王忠诚主编.王忠诚神经外科学[M].武汉：湖北科学技术出版社,2005
[2]Mao X G, Zhang X, Zhen H N. Progress on Potential strategies to target brain tumor stem cells[J].Cell Mol Neurobiol,2009,29(2):141-155
[3]Hegedus B, Dasgupta B, Shin J E, et al. Neurofibromatosis-1 regulates neuronal and glial cell differentiation from Neuroglial Progenitors in vivo by both cAMP and Ras-dependent mechanisms. Cell Stem Cell 007:1:443-457
[4]Singec 1, Knoth R, Meyer RP,et al.Defining the actual sensitivity and specificity Of the neurosphere assay in stem cell biology.Nat Methods 2006;3:801-6
[5]Ignatova TN, Kukekov VG, Laywell ED,et al Human cortical glial tumors contain neural stem-like cells expressing astroglial and neural markers in vitro.Glia 2002;39:193-206
[6]Hambardzumyan D,Squatrito M, Holland E C.Radiation resistance and Stem-like cells in brain tumors[J].Cancer Cell,2006,10(6):454-456.
[7]Pellegatta S, Poliani PL, Como D,et al. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.Cancer Res,2006,66(21):10247-10252.
[8]Bernard DJ, Courjal F, Maurizis JC, et al. Effect of epidermal growth factor in HLA class I and class II transcription and protein pression in human breast adenoeareinoma cell lines. Br J Cancer 1992,66:88-92
[9]康德智,苏东辉等.高表达Hsp70鼠脑胶质瘤细胞瘤苗的体外抗瘤作用[J].中华神经医学杂志,2003.2(1)：52-56.
[10]Heimberger AB,Sampson JH.The PEPvⅢ-KLH(CDX-110) vaccine in Gliob-lastoma multiforme Patients. Expert OPin Biol Ther.2009:9(8):1087-1098
[11]Roszman TL, Brooks WH,Elliott LH.Immunobiology of Primary in traeranial tumors.VI.Suppressor cell function and lectin-binding lymphocyte Subpopulations in patients with cerebral tumors. Cancer 1982 Oct 1:50(7):1273-9.
[12]Jordan C T, Guzman M L, Noble M. Cancer stem cells[J].N Engl J Med,2006,355 (12) :1253-1261.
[13]Xie Z.Brain tumor stem cells [J]. Neurochem Res,2009,34(12):2055
[14]Bonnet D,Diek JE.Human acute myeloid leukemia is organized as A hierarehy that originates from a primitive hematopoietic cell[J].Nature Med,1997,3(7):730-737.
[15]Al-Hajj M, Wicha MS, Benito HA, et al. Prospcetive Identification of tumorigenic breast cancer cells[J].Proc Natl Acad Sci USA,2003,100(7):3983-3988.
[16]Singh S K, Hawkins C, Clarke I D, et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396-401
[17]Veseovi AL, Galli R, Reynolde BA, et al. Brain tumor stem cell[J]. Nature,2006,6(17):425-436.
[18]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133+cancer stem cells in glioblastoma [J]. Mol Cancer,2006,5:67.
[19]Bao S,Wu Q,Melendon R E,et al.Glioma stem cells promote radioresistance by Preferential activation of the DNA damageresponse[J].Nature,2006,444(7120):756-760.
[20]Brewer GJ Isolation and culture of adult rat hippocampal neurons [J]. Neurosci, Methods,1997,71(2):143-155
[21]Groszer M, Erickson R, Scripture-Adams DD, et al. Negative regulation of neural stem Progenitor cell Proliferation by the Pten tumor suppressor gene in vivo[J]. Science,2001 294(5549):2186-189.
[22]Li C, Heidt DG, Daletha P, Burant CF, et al Identification of Pancreatic caneer Stemcells.Cancer Res 2007;67:1030-1037
[23]Singh S K, Hawkins C, Clarke I D, et al. Identification of human brain tumour initiating cells[J]. Nature,2004,432(7015):396-401
[24]Wan F, Zhang S, xie R, et al. The utility and limitations of Neurosphere assay, CD133 immunophenotyping and side population assay in glioma stem cell Research[J].Brain pathol,2010,20(5):877-889.
[25]CamPos B,Herold-MendeC C.Insight into the complex regulation of CD133 in glioma [J].Int J Cancer,2011,128(3):501-510.
[26]张全斌,黄强,兰青.脑肿瘤干细胞的热点问题及研究进展[J].中华医学杂志,2005(26)：1868-1870.
[27]Figdor CG, de Vries IJ, Lesterhuis WJ, et al. Dendritic cell immunotherapy:mapping the way.Nat Med 2004; 10:475-80.
[1]孟凡东,隋承光,王晓华等.体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究[J].中国年学杂志,2006;26(6)：818-820
[2]Stupp R, Hegi M E. Targeting brain-tumor stem cells [J]. Nat Biotechnol, 2007,25(2):193-194
[3]刘福生,金贵善,历俊华等.树突状细胞与反义TGF-β1基因修饰的胶质瘤细胞融合瘤苗治疗脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：47-50
[4]俞建波,冯志仙,詹仁雅,童鹰,马跃辉,潘剑威 RNA 致敏树突状细胞治疗胶质瘤的实验研究浙江大学学报(医学版),Journal of Zhejiang University(Medical Sciences),2007,36(6)92-97
[5]Steinman RM, Cohn ZA. Identification of a novel cell type in Peripheral lymphoid organs of mice. MorPhology, quantitation, tisSued distribution [J].J Exp Med,1973,137(5):1142-1162.
[6]Walker PR,Calzascia T,Schnuriger V, et al. The brain parenchyma is permissive for full antitumor CTL effector function,even in the absence of CD4 T cells. [J].Immunol.2000;165:3128-3135
[7]Brossart P,Wirths S,Brugger W,et al. Dendritic cells in cancer vaccines[J] ExP Hematol,2001,29(11):1247-1255.
[8]Dendritic cells pulsed with Total tumor RNA for activation NK-like T cells against glioblastoma multiforme[J].J Neurooncol,2005,75(2):111-118
[9]Berney C, Herren S, Power CA, et al. A member of the dendritic cell family that enters B cell follicles and stimulates Primary antibody responses identified by a mannose receptor fusion Protein [J] J ExP Med,1999,190(6):851-860.
[10]Kiertscher SM, Luo J, Dubinett SM, et al.Tumors promote altered maturation and early apoprosis of monocyte derived dendritic cells[J].J Immununol,2001,164(7):1269-1277.
[11]Steinman RM, Turley S, Mellmanl,et al.The induction of tolerance by dendritic cells tha thave captured apoptotic cells. J.Exp.Med.2000; 191:411-416
[12]Hagihara M, Li C, Gansuvd B, et al. Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma [J].Immunol Methods,2001,253(1-2):45-55.
[13]Sehendel DJ. Dendritic cell vaccine strategies for renal cell careinoma[J).Expert opin Biol Ther,2007,7(2):221-232.
[14]Koido S, Hara E, Homma S, et al. Dendritic/tumor fusion cell-based vaccination against cancer[J].Arch Immunol Ther Exp(Warsz),2007,55(5):281-287.
[15]Ardavin C,Amigorena S, Reis E S C. Dendritic cells:immunobiology and cancer immunotherany[J].Immunity,2004,20(1):17-23.
[16]Holland EC, Celestino J, Dai C,et al. Combined activation of Ras and Akt in neural progenitors Induces glioblastoma formation in mice[J]. Nat.Genet.2000,25(l):55-57
[17]冯志华,王全楚主编.新概念疫苗[M].北京：人民卫生出版社,2004.
[18]Banchereau J, Briere F, Caux C, et al.Immunobiology of dendritic cells[J].Annu Rev 1 mmunol,2000,18:767-811.
[19]Liau LM,Prins RM,Kiertscher SM, et al.Dendritic cell vaccination in glioblastoma patients induces systemic and intraecranial T-cell responses modulated by the local central nervous system tumor microenvironment. Clin Cancer Res 2005:11(15):5515-25.
[20]王东海,李新钢,曲迅,等.树突状细胞融合瘤苗抗胶质瘤的实验研究.中华神经外科杂志,2006,22：699-701.
[21]Yamanaka R, Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase IPII tria [J]. Clin CancerRes,2005; 11 (11):4160-4167
[22]Ashley D M, Faiola B, Nair S, et al. Bone marrow-Generated dendritic cells pulsed with tumor extracts or tumor RNA induce antitumor immunity against central nervous system tumors[J]J Exp Med,1997,186(7):1177-1182
[23]Heimberger AB,Abou-Ghazal M, Reina-Ortiz C, et al. Incidence and Prognostic Impact of Fox P3+regulatory T cells in human gliomas.Clin Cancer Res 2008 15;14(16):5166-72
[24]Pellegatta S, Poliani PL, Como D,et al. Neurospheres enriched in cancer stem-like cells are highly effective in eliciting a dendritic cell-mediated immune response against malignant gliomas.Cancer Res,2006,66(21):10247-10252.
[1]Jeremias I, Steiner HH, BennerA, et al. Cell death induction by betulinic acid, ceramide and TRAIL in primary glioblastoma multiforme cells [J]. Acta Neurochir,2004; 146 (7):721-729
[2]姜政,江玉泉,李新钢,等.人脑胶质瘤细胞原代培养实验模型的构建[J].中华肿瘤防治杂志,2006；13(10)：744-747
[3]陈忠平周旺宁我国胶质瘤诊断治疗现状和努力方向 中国肿瘤,Bulletin of Chinese Cancer,2005,14, (2) 78-81
[4]窦长武；树突状细胞介导的抗脑胶质瘤的免疫治疗进展 内蒙古医学院学报,Acta Academiae Medicinae Neimongol,2007,29 (6) 452-455
[5]Lendah U, Zimmerman LB, Mckay RD. CNS stem cell express a new class of intermediate filament protein[J]. Cell,1990,60(4):585-595
[6]Recht L, Jang T, Savarese T, et al. Neural stem cells and neuIo-on-eulogy:quo vadis[J]. J Cell Biochem,2003,88(1):11-19.
[7]于春泳,魏学忠,宋振全.成熟大鼠脑损伤后神经干细胞的表达与增殖[J].中国临床康复,2002,6(2)：1744-1745.
[8]Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell [J]. Nat Med,1997,3(7):730-737.
[9]Matsui W, Huff CA, Wang Q, et al. Characterization of clonogenic multiple myeloma cells[J]. Blood,2004,103(6):2332-2336.
[10]AI-Hajj M, Wicha MS, Benito-Hernandez A, et ol. Prospective identification of tumorigenic breast cancer cells[J]. Proc Natl Acad Sci USA.2003,100(7): 3547-3549.
[11]Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stem cells[J]. Nature,2001,404(6859):105-111.
[12]Ignatova TN, Kukekov VG, Laywell ED, et al. Human cOf tical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro [J]. Glia, 2002,393; 193-206.
[13]Singh SK, Clarke ID, Terasaki M, et ol. Identification of cancer stem cell in human brain tumors [J]. Cancer Res 2003; 63181:5821-5828.
[14]Hemmati HD, Nakano I, Lazareff JA, Cancerous stem cells can arise from pediatric brain tumors[-[J]. Proc Natl Acad Sci USA,2003,100(25):15178-15183.
[15]Yi L, Zhou ZH, Ping YF, et al. Isolation and characterization from primary human anaplastic oligoastroeytoma [J]. Modern Pathology,2007,20:1061-1068.
[16]Kondo T, Setoguchi T, Taga T. Persistence of a small sub-population of cancer stein-like ceils in the C6 glioma cell line[J]. Proct Natl Acad Sci USA,2004,101(3): 781-786.
[17]Ghods AJ, Irvin D, IAu G, et cd. Spheres isolated from 9L gliosarcoma rat cell line possess chemoresistant and aggressive cancer stem-like cells [J]. Stem Cells,2007, 25(7):1645-1653.
[18]Yu SC, Ping YF, Yi L, et al. Isolation and characterization of cancer stem cells from U87[J]. Cancer Letters,2008,265:124-134.
[19]STROJNIK T, ROSLAND G V, SAKARIASSEN P O, et a 1. Neural stem cell markers, nestin, and musashi proteins, in the progression of hum-an glioma: correlation of nestin with prognosis of patient s-rvival[J]. Surg Neurol,2007,68 (2): 133-144.
[20]SINGH S CLARKE ID, HIDE T, et al. Cancer stem cells in nervous system tumors [J]. Oncogene,2004,23(43):7267-7273
[21]BERGER F, GAY E, PELLETIER L, et al. Development of gliomas:potential role of asymmetrical cell division of neural stem cells [J]. lancet Oncol,2004,5(8):511-5 14.
[22]黄强,董军,朱玉德,等.人脑胶质瘤组织中分离与培养肿瘤干细胞[J].中华肿瘤杂志,2006,28(5)：331-333.
[23]Raso A, Negri F, Gregorio A, et al. Successful isolation and long-term establishment of a cell line with stem cell, like features from all anaplastic medulloblastoma[J J. Neuropathol Appl Neurobiol,2008,34(3):306-315.
[24]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133 cancer stem cells in glioblastoma[J]. Mol Cancer, 2006,5:67.
[25]周志华,易良,卞修武,等.人恶性胶质瘤细胞株U251三种克隆的分化特性和胶质瘤干细胞的初步鉴定[J].中华神经外科杂志,2008,24(9)：658-662.
[26]Yuan XP, Curlin J, Xiong YZ, et al. Isolation of cancer stem cells from adult glioblastoma multiform [J]. Oncogene,
[27]季晓燕,黄强,董军等,脑肿瘤干细胞体外分化的形态标志物及细胞增殖动力学特征[J].中华医学杂志,2006,86(23)：1604-1609.
[28]Barnett S C, Robertson L, Graham D, Allan D, and Rampling R Oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells transform ed with c-myc an d H-ras form high-grade glioma after stereotactic injection into the rat [J]. Carcinogenesis,1998,19(9):1529-1537.
[29]Barnett S C, Robertson L, Graham D, Allan D, and Rampling R Oligodendrocyte-type-2 astrocyte (0-2A) progenitor cells transform ed with c-myc an d H-ras form high-grade glioma after stereotactic injecfion into the rat brain[J] Carcinogenesis,1998,19(9):1529-1537.
[30]Beier D, Hau P, Proescholdt M. CD133 and CD133-glioblastoma. derived cancer stem ceUs show differential growth characteristics and molecular profiles[J]. Cancer Res,2007,67(9):4010-4015.
[31]Wechsler-Reya RJ. Analysis of gene expression in the normal and malignant cerebellum [J]. Recent Prog Horm Res,2003; 58:227-248.
[32]Lubensky IA, Vortmeyer AO, Kim S, et ol. Identification Of tumor precursor cells in the brains of primates with radia. Tion-induced de novo glioblastoma multiforme[J]. Cell Cycle,2006; 5(4):452-456.
[33]Aboody KS, Brown A, Rainov NG, et ol. Neural stem cells display extensive tropism for pathology in adult brain:evidence from intracranial gliomas[J]. Proc Natl Acad Sci US-A.2000; 97(23):12846-12851.
[34]Hanahan D, Weinbery RA. The hallmarks of cancer J.Cell,20000,100;57-70
[35]李茗初,邓永文,伍军等.髓母细胞瘤中脑肿瘤干细胞的分离培养及鉴定[J].癌症,2006；25(2)：241-246.
[36]Bschoo RM,Maher EA, Ligon KL, et al Epidermal groeth factor receptor and Ink4a/Aft [J] Cancer cell 2002,1,(3):269-277
[37]Holland EC. Gliomagenesis:genetic alterations and mouse models[J]. Nat Rev Genet, 2001; 2(1):120-129.
[38]Nagakawa M,Kpyangagi M.,et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts J Nat Biotechnol 2008,26(1):101-106
[39]Hope KJ,Jin LQ, Dick JE, et al. Acute myeloid Leukemia originate from a hierarchy of leukemic stem cell classess that differ in self-renewal capccity J Nature Imunol,2004,5:738-743
[40]Joo KM, Kim SY, Jin X, et al. Clinical and biological implications of CDI33-positive and CDI33-negative cells in glinblastomas[J]. Lab Invest,2008,88(8):808-815.
[41]Zheng X, Shen G, Yang X, et al. Most C6 cells are cancer stem cells:evidence from clonal and population analyses[J]. Cancer Res,2007,67(8):3691-3697.
[42]Wang J, Sakariassen PQ, Tsinkalovsky O, et al CD 133 negative glioma cells from tumors in nude rats and give rise to CD133 positive cells[J]. Int J Cancer,2008, 122(4):761-768.
[43]陈骅,黄强.胶质瘤干细胞研究的新观点[J].中国神经肿瘤杂志,2008,1(6)：1-4.
[44]Shervington A, Lu C. Expression of multidrug resistance genes in normal and cancer stem cells[J]. Cancer Invest,2008,26(5):535542
[45]Liu G, Yuan X, Zeng Z, et al. Analysis of gene expression and chemoresistance of CD 133 cancer stem cells in glioblastoma[J]. Mol Cancer,2006,5:67.
[46]Lu C, Sheiwington A. Chemoresistance in gliomas[J]. Mol Cell Biochem, 2008.312(1-2):71-80
[47]Bi CL, Fang JS, Chen FH, et al. Chemoresistance of CD133(+) tumOr stern cells from human brain glioma[J]. Zhong Nan Da Xue Xue Bao Yi Xue Ban,2007,32(4): 568-573.
[48]Beier D, Ri-hrl S, Pillai DR, et al. Temozolomide preferentially depletes cancer stem cells in glioblastoma[J]. Cancer Res,2008,68(14):5706-5715.
[49]Bao S, Wu Q, McLendon RE, et al. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response [J]. Nature,2006,444(7120): 756-760.
[50]Blazek ER. Foutch JL. Maki G. Daoy medulloblastoma cells that express CD133 are radioresistant relative to CD133-cells, and the CD133+sector is enlarged by hypoxia[J]. Int J Radiat Oncol Biol Phys,2007,67(1):1-5.
[51]Hambardzumyan D, Becher OJ, Rosenblum MK, et al. PI3 K pathway regulates survival of cancer stenl cells residing in the perivascular niche following radiation in medulloblastoma in vivo[J]. Genes Dev,2008,22(4):436-448.
[51]KleinRS,LinE,ZhangB,etal.Neuronal CXCL10 directs CD8+T-cell recruitment and control of West Nile virus encephalitis [J]. JVirol,2005,79:11457-11466.
[52]SeoDR, KimKY, LeeYB.Interleukin-10 expression in lipopolysaccharide-activated microglia is mediated by extracellular ATP in an autocrine fashion [J] Neuroreport,2004,15:1157-1161.
[53]Tsugawa T,KuwashimaN,SatoH,etal.Sequential de-livery of interferon-a gene and DCs to intracranial gliomas promotes an effective antitumor response[J].Gene Ther,2004,11:1551-1518.
[54]刘福生,金贵善,历俊华,等.树突状细胞与反义TGF-β1基因修饰的胶质瘤细胞融合瘤苗治疗脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：47-50
[55]俞建波,冯志仙,詹仁雅,童鹰,马跃辉,潘剑威RNA致敏树突状细胞治疗胶质 瘤的实验研究浙江大学学报(医学版),Journal of Zhejiang University(Medical Sciences),2007,36(6)92-97
[56]YANG B Y, WU CY。The effect of vaccine adjuvants such as IL.12and CpG to CD4+ Tcells induced specific immune response in vivo[J].Chin J Immunol,2005,11: 803-807.
[57]孟凡东,隋承光,王晓华等.体外培养的树突状细胞与细胞因子诱导的杀伤细胞相互作用的研究[J].中国年学杂志,2006；26(6)：818-820
[58]Held FJ,Lutjohann B,Ungerfroren H, et al. Interaction of transforming growth factor-beta TGF-beta and epidermal growth factor EGF in human glioma cells[J]. J Neurooncol,2003; 632:117
[59]Yamanaka R,Homma J, Yajima N, et al. Clinical evaluation of dendritic cell vaccination for patients with recurrent glioma:results of a clinical phase IPII tria [J]. Clin CancerRes,2005; 11 (11):4160-4167
[60]宫安静,孟庆海,朱湘华,等.树突状细胞诱导的CTLs与CIK及LAK细胞对神经胶质瘤杀伤作用比较[J].青岛大学医学院学报,2007；43(2)：159-161
[61]于音,赵刚多基因联合在胶质瘤治疗中的应用进展山东医药,Shandong Medical Journal,2007年33期
[62]Parajuli P, Mathupala S, Sloan AE. Systemic comparison of dendritic cell-based immunotherapeutic strategies for malignant gliomas:in vitro induction of cytolytic and natural killer-like T cells. Neurosurgery,2004,55(5):1194-1204
[63]Saito R, Mizuno M, Nakahara N, et al. Vaccination with tumor cell lysate-pulsed dendritic cells augments the effect of IFN-beta gene therapy for malignant glioma in an experimental mouse intracranial glioma. Int J Cancer,2004,111(5):777-82
[64]孟庆海,薄勇力,李洛等.树突状细胞疫苗治疗大鼠脑胶质瘤的实验研究[J].中华神经外科杂志,2006；22(1)：55-58
[19]Yu JS,Wheeler CJ, Zeltzer PM. et al. Vaccination of malignant glioma patients with peptide-pulsed dendritcells elicits systemic cytotoxicity and intracranial T-cell infiltration[J]. CancerRes,2001;613:842-847
[65]赵丽波,李才,周维国树突状细胞负载的肿瘤抗原肽疫苗抗脑胶质瘤作用的实验研究中国实验诊断学,[J]. Chinese Journal of Laboratory Diagnosis, 2009.01(1):112-114
[66]Akasaki Y, Kikuchi T, Homma S, et al. Antitumor effect of im-munizations with fusion of dendritic and glioma cells in a mouse brian tumor model. J Immunother,2001, 24(2):106-113.
[67]Kjaergaard J,WangLX, Kuriyama H, et al. Active immunotherapy for advanced intracranial murine tumors by using dendritic cell-tumor cell fusion vaccines [J]. J Neurosurg,2005;103 (1):156-164
[68]RyuyaYamanaka Cell-and peptide-based immunotherapeutic approaches for glioma
[69]Wilm Mae, DC vaccination with anti-CD25 treatment leads to long-term immunity against experimental glioma, Neuro Oncol 2009, DOI:10.1215/15228517-2009-004
[70]张晓实树突细胞疫苗治疗脑胶质瘤的研究进展中国神经肿瘤杂志,Chinese Journal of Neuro-Oncology,2007; 5 (3) 149-151
[71]Ludewig B, Ochsenbein AF, Odermatt B, et al. Immunotherapy with dendritic cells directed against tumor antigens shared with normal host cells results in severe autoimmune disease. [J]Exp Med,2000,191 (5):795-804.