靶向脑胶质瘤干细胞的树突状细胞疫苗治疗恶性脑胶质瘤的体外实验研究

目的 研究应用脑胶质瘤干细胞抗原制备的树突状细胞(DC)疫苗对胶质瘤细胞的杀伤作用.方法 反复冻融法获得源于胶质瘤细胞系U251中的肿瘤干细胞的胞溶物,和源于小鼠间充质干细胞的DC共培养,获得胶质瘤干细胞疫苗.流式细胞仪检测疫苗表面标志变化;CCK-8法检测其促T细胞增殖能力以及对胶质瘤细胞的靶向杀伤作用;ELISA法检测培养基中干扰素-γ的含量.用热处理U251细胞制备的DC疫苗作为对照组.结果 与对照组相比,脑胶质瘤干细胞疫苗的CD80、CD86、CD11c和MHC Ⅱ等表面标志表达显著提高,具有更强的促T细胞增殖能力(P＜0.01),对U251细胞特异性靶向杀伤率随效靶比的提高而增加,最高为(78.508±4.156)％,相应的干扰素-γ分泌水平也达到最高.结论 胶质瘤干细胞疫苗能更有效地诱导特异性细胞毒性T细胞,表现出更强的抗肿瘤特性.     

凋亡肿瘤细胞抗原致敏的树突状细胞瘤苗治疗大鼠颅内胶质瘤的实验研究

目的观察凋亡肿瘤细胞抗原致敏的树突状细胞(dendriticcell,DC)瘤苗对颅内胶质瘤免疫治疗的效果。方法通过立体定向接种建立Wistar大鼠C6胶质瘤动物模型;从大鼠骨髓分离DC前体细胞,经重组大鼠粒细胞巨噬细胞集落刺激因子(rrGM-CSF) 白细胞介素4(rrIL-4)诱导培养、扩增获得功能性DCs;DCs经采用热诱导凋亡的C6胶质瘤细胞体外致敏后皮下回输荷瘤大鼠体内,1次/周,共5次。观察荷瘤大鼠的存活期,MRI观察颅内肿瘤生长情况,循环血中CD8 T淋巴细胞水平及体外细胞毒反应、增殖反应均以流式细胞仪检测。结果经过治疗的荷瘤大鼠生存期明显延长,MRI显示实验组大鼠肿瘤被明显抑制,外周血中CD8 T淋巴细胞比例增加,体外细胞毒试验提示经凋亡肿瘤细胞抗原致敏的DC瘤苗可以诱导针对C6胶质瘤的特异性细胞毒性T淋巴细胞,并且未观察到自身免疫反应的发生。结论经凋亡肿瘤细胞抗原致敏的DC瘤苗对于颅内胶质瘤是一种安全有效的治疗方法。     

树突状细胞制备脑胶质瘤疫苗的体外研究

目的　通过体外实验探讨应用树突状细胞制备的肿瘤疫苗抗脑胶质瘤的作用机制。方法　培养大鼠树突状细胞,冻融法制备胶质瘤抗原,以肿瘤抗原致敏树突状细胞而制备胶质瘤疫苗。将实验动物分为4组,分别将胶质瘤疫苗、树突状细胞、生理盐水注入正常大鼠体内,到达预定时间取出大鼠脾脏,MTT法检测大鼠淋巴细胞活性。结果　1次输入胶质瘤疫苗的正常大鼠的淋巴细胞对肿瘤细胞的杀伤活性明显低于3次输入胶质瘤疫苗的正常大鼠;比较输入培养8d的树突状细胞和输入生理盐水的大鼠,其淋巴细胞对肿瘤细胞的杀伤活性无统计学差异。结论　应用树突状细胞制备胶质瘤疫苗可明显激活体内抗肿瘤免疫反应。     

IL-13Rα2致敏的DC-CTL对人胶质瘤干细胞的体外杀伤效应

目的 比较IL - 13Rα2致敏的DC - CTL对人胶质瘤干细胞和普通胶质瘤细胞的体外杀伤效应.方法 用神经干细胞无血清培养法培养出U251胶质瘤干细胞并进行免疫荧光鉴定,GM- CSF、IL-4体外诱导成树突状细胞(DCs),与人工合成的IL - 13Rα2(345-354)多肽孵育后再激活T淋巴细胞,CFDA - SE/PI双荧光染料标记的流式细胞计数法检测其对U251胶质瘤干细胞和普通U251胶质瘤细胞的体外杀伤作用.结果IL - 13Rα2(345-354)抗原肽致敏的DC - CTL对U251胶质瘤干细胞的杀伤率为(18.59±1.42)％,高于未用抗原肽致敏的DC - CTL及CIK对胶质瘤干细胞的杀伤率,其杀伤率分别为(10.35±0.98)％和(7.15±0.58)％,差异具有统计学意义(P＜0.001).同时,IL- 13Rα2(345-354)抗原肽致敏的DC - CTL对U251胶质瘤干细胞的杀伤率也高于其对普通U251细胞(11.53±0.65)％的杀伤率(P=0.001).结论 IL - 13 Rα2(345-354)致敏的DC - CTL在体外对胶质瘤于细胞具有杀伤作用,且杀伤率高于对普通的胶质瘤细胞.     

树突状细胞疫苗治疗G422胶质母细胞瘤的实验研究

目的研究G422肿瘤细胞RNA体外冲击致敏的树突状细胞(DC)回输诱导体内产生保护性免疫反应及对颅内荷瘤小鼠的免疫治疗效应,探讨以树突状细胞为基础的疫苗对脑胶质瘤治疗的可行性.方法提取G422胶质母细胞瘤RNA或肿瘤提取物冲击致敏DC制成疫苗,分别进行免疫保护和免疫治疗的体内实验,以及细胞毒性T淋巴细胞(CTL)体外诱导及活性检测,并与PBS、未经致敏的DC、G422肿瘤细胞RNA组进行对照.疫苗能诱导产生针对G422肿瘤抗原特异性CTL,差异具有非常显著性(P＜0.01),接种疫苗后的小鼠能抵抗G422的再次攻击(P＜0.01),荷瘤小鼠接种疫苗后生存期较对照组延长(P＜0.05).结果疫苗能诱导产生针对G422肿瘤抗原特异性CTL,差异具有非常显著性(P＜0.01),接种疫苗后的小鼠能抵抗G422的再次攻击(P＜0.01),荷瘤小鼠接种疫苗后生存期较对照组延长(P＜0.05).结论肿瘤RNA或肿瘤提取物冲击致敏DC能诱导小鼠产生抗原特异性CTL,激活抗肿瘤T细胞,具有免疫保护和免疫治疗作用.该实验为DC疫苗免疫治疗胶质瘤提供了实验基础.     

肿瘤RNA冲击致敏树突状细胞治疗G422胶质母细胞瘤的实验研究

目的 研究G422肿瘤细胞RNA体外冲击致敏的树突状细胞（DC）回输诱导体内产生保护性免疫反应及对颅内荷瘤小鼠的免疫治疗效应，探讨以DC为基础的疫苗对脑胶质瘤治疗的可行性。方法 提取G422胶质母细胞瘤RNA冲击致敏DC制成疫苗，分别进行免疫保护和免疫治疗的体内实验，以及细胞毒性T淋巴（CTL）体外诱导及活性检测，并与PBS，未经致敏的DC，G422肿瘤细胞RNA组进行对照。结果 疫苗能诱导产生针对G422肿瘤抗原特异性CTL，具有非常显著性差异（P＜0．01），接种疫苗后的小鼠能抵抗G422的再次攻击（P＜0．01），疫苗组生存期较对照组延长（P＜0．05）。结论 肿瘤RNA体外冲击致敏DC，然后将之回输免疫接种至颅内荷瘤小鼠能显著地诱导机体产生抗原特异性CTL，激活抗肿瘤T细胞，具有免疫保护和免疫治疗作用，为DC疫苗免疫治疗胶质瘤提供了实验基础及理论依据。     

自体肿瘤树突状融合细胞体外诱导抗人脑胶质瘤活性的研究

目的本实验使用人脑胶质瘤细胞和同一患者的外周血单个核细胞(PBMC)诱导的树突状细胞(DC)融合来制备DC-肿瘤融合细胞(FC),并研究其体外诱导抗胶质瘤的活性。方法采用PEG法将来自胶质瘤患者的肿瘤细胞和其自体DC进行融合,采用混合淋巴细胞反应和乳酸脱氢酶法(LDH)释放法来分别评价FC刺激T淋巴细胞增殖能力和FC致敏的T淋巴细胞对相同患者胶质瘤细胞的杀伤能力。结果建立了DC-人脑胶质瘤融合细胞(FC)的制备方法,通过荧光双染色证明FC具有胶质瘤细胞和DC细胞的双重标记,经过1.5Gy照射的FC致敏患者外周血T细胞,对自体胶质瘤细胞的杀伤明显高于仅用单纯DC或自体胶质瘤细胞致敏的T细胞(P<0.01),而且杀伤能力随E/T率的增加而由28%上升到90%,但对乳腺癌细胞MCF7的杀伤仅在10%左右,显著低于对自体胶质瘤细胞的杀伤(P<0.01);同时致敏的T细胞在负载患者胶质瘤细胞裂解物(lysate)的自体DC的刺激下引起的增殖高于仅用单纯DC或自体胶质瘤细胞致敏的T细胞,并具有统计学意义(P<0.05);对于已经致敏的T淋巴细胞,负载lysate的自体DC所引起的T细胞增殖明显高于自体DC或lysate的作用(P<0.01)。结论本实验所制备的FC经过1.5Gy照射后,可以有效致敏患者外周血T细胞,并在负载抗原的DC的刺激下可以在体外扩增,致敏的患者外周血T淋巴细胞是可以进行特异性杀伤的CTL,杀伤能力随效靶比增高而上升。     

沙利度胺联合替莫唑胺杀伤U251胶质瘤细胞机制的体外研究

目的 对沙利度胺联合替莫唑胺杀伤U251胶质瘤细胞的机制进行体外研究,为制订沙利度胺与替莫唑胺联合化疗方案提供理论依据.方法 经体外培养的人胶质瘤细胞系U251分别接受替莫唑胺(100 μmol/L)、沙利度胺(100 μg/L)、替莫唑胺与沙利度胺联合治疗,噻唑监(MTT)法检测不同抗肿瘤药物处理组肿瘤细胞增殖活性;流式细胞术分析细胞增殖周期;检测经吖啶橙标记的酸性囊性细胞器数目;原位末端标记(TUNEL)法观察肿瘤细胞凋亡情况;Western blotting法榆测肿瘤自噬及凋亡相关蛋白表达变化.结果 与替莫唑胺和沙利度胺单药治疗相比,替莫唑胺与沙利度胺联合治疗对U251细胞生长的抑制更为明显(均P=0.000).且可诱导肿瘤细胞周期阻滞于G0～G1期,以及发生凋亡和自噬.两药联合治疗后,U251细胞微管相关蛋白1轻链3和Caspase-3表达水平高于替莫唑胺组和沙利度胺组(均P=0.000).结论 沙利度胺联合替莫唑胺治疗U251细胞可以上调自噬及凋亡相关基因表达水平.同时诱导凋亡及自噬性死亡,从而达到对U251胶质瘤细胞的杀伤作用.     

树突状细胞瘤苗抗脑胶质细胞瘤作用体外实验研究

目的通过体外实验探讨应用树突状细胞制备的肿瘤疫苗抗脑胶质瘤的作用机制.方法培养大鼠树突状细胞,冻融法制备胶质瘤抗原,以肿瘤抗原致敏树突状细胞而制备胶质瘤疫苗.将实验动物分为4组,分别将胶质瘤疫苗、树突状细胞、生理盐水注入正常大鼠体内,到达预定时间取出大鼠脾脏,四噻唑蓝(MTT)法检测大鼠淋巴细胞活性.结果一次输入胶质瘤疫苗的正常大鼠的淋巴细胞对肿瘤细胞的杀伤活性明显低于3次输入胶质瘤疫苗的正常大鼠;比较输入培养8 d的树突状细胞和输入生理盐水的大鼠,其淋巴细胞对肿瘤细胞的杀伤活性无统计学差异.结论应用树突状细胞制备胶质瘤疫苗可明显激活体内抗肿瘤免疫反应.     

胶质瘤光动力疫苗的体外细胞毒性实验研究

目的制备胶质瘤光动力疫苗并检测体外抗肿瘤效应。方法采用4种不同方法制备胶质瘤疫苗。冻融疫苗:冻融法制备人胶质瘤细胞U251抗原,呈递给树突状细胞(DC)制备疫苗。热休克疫苗:热休克法处理人胶质瘤细胞U251并提取抗原,呈递给DC制备疫苗。光动力全细胞抗原疫苗:光动力法提取人胶质瘤细胞U251全细胞抗原并呈递给DC制备疫苗。光动力洗脱抗原疫苗:光动力法处理人胶质瘤细胞U251后,再用弱酸洗脱法提取活细胞表面抗原,呈递给DC制备疫苗。将上述4种不同方法制备的疫苗在体外与自体淋巴细胞以及靶细胞U251反应,然后用MTT法检测靶细胞凋亡率及坏死率,比较4种疫苗的体外抗肿瘤细胞效应。结果冻融疫苗、热休克疫苗、光动力全细胞抗原疫苗和光动力洗脱抗原疫苗的靶细胞死亡率分别是:(10.02±0.32)%、(18.34±0.46)%、(35.56±1.75)%和(54.45±2.14)%,4组疫苗抗肿瘤细胞效应是光动力洗脱抗原疫苗光动力全细胞抗原疫苗热休克疫苗冻融疫苗。结论胶质瘤光动力洗脱抗原疫苗是一种具有良好体外抗肿瘤效应的新型疫苗。     

神经干细胞与脑胶质瘤干细胞☆

所有的肿瘤组织并不是由均一的肿瘤细胞所组成的,不同的细胞具有不同的增殖、浸润和转移能力,亦即肿瘤的异质性。其中存在少数担当着干细胞角色的肿瘤细胞,具有干细胞的基本特性,包括自我更新能力、无限的增殖能力和多向分化潜能,为肿瘤干细胞。神经干细胞具有很强的自我更新机制,获得较少突变即有可能恶性转化,而且干细胞存活时间较长,这意味着干细胞比成熟细胞发生细胞复制的错误几率更大,因外界环境的刺激而发生突变的机会更多,最终形成脑胶质瘤干细胞,同时调节神经干细胞增殖和自我更新的基因在脑胶质瘤的脑胶质瘤干细胞中也表达,这也是支持神经干细胞是脑胶质瘤干细胞来源的;也有推测认为它可能起源于已分化的细胞,由这些细胞突变发生去分化得来,并通过基因突变而获得了干细胞自我更新的特性,从而形成脑胶质瘤干细胞。通过探讨神经干细胞与脑胶质瘤干细胞,为脑胶质瘤的治疗提供依据。     

Quite amusing stem cells:Muse cells

<正>Stem cells may be the future of therapeutics for stroke due to their regenerative and immunomodulatory capabilities.Major barriers faced when employing stem cells,however,include faulty migration,low cell survival,and diminished proliferation.M ultilineage-differentiating stress ensuring (Muse) cells,a subset of mesenchymal stem cells,overcome these barriers.Muse cells aid in neuroregeneration,have immense regenerative potential,and are pluripotent,non-tumorigenic,and immunomodulatory.I...     

Oligodendroglioma-like cells (clear cells) in ependymoma

A brain tumor of a 22-year-old man was composed mostly of round cells with perinuclear halos (clear cells), forming clusters intersected by small blood vessels. In some areas, the tumor cells showed perivascular arrangement and epithelial pattern. Phosphotungstic-acid hematoxylin stain and immunoperoxidase stain for glial fibrillary acidic protein (GFAP) technique failed to stain the clear cells. Electron microscopy of the clear cells revealed them to be classical ependymoma cells with well developed intercellular junctions, microvilli and cilia. As no reporters in the past showed the evidence to clarify the nature of the clear cells, this case is considered a good example to support the viewpoint that the clear cells (oligodendroglioma-like cells) commonly observed in ependymomas are in reality ependymoma cells. It is stressed that the diagnosis of "mixed glioma" or "oligoependymoma" should be made with sufficient caution despite the recent advances of GFAP technique.     

神经干细胞诱导骨髓基质细胞向神经元样细胞分化

目的：研究骨髓基质细胞向神经元样细胞分化的条件。方法：神经十细胞诱导骨髓基质细胞向神经元样细胞分化，免疫细胞化学方法对分化的和未分化的细胞进行鉴定。结果：神经于细胞可诱导骨髓基质细胞向神经元样细胞分化，分化的细胞表达神经元的标志物神经元特异性烯醇化酶(NSE)。结论：骨髓组织中存在能分化为神经元的于细胞，可能成为中枢神经系统自体移植的于细胞的来源。     

Transplanted human bone marrow cells generate new brain cells

Multiple studies have reported that adult cells of bone marrow origin can differentiate into muscle, skin, liver, lung, epithelial cells, and neurons. To determine whether such cells might produce neurons and other cells in the human brain, we examined paraffin sections from female patients who had received bone marrow transplants from male donors. Y-chromosomes were labeled using autoradiography and fluorescent in situ hybridization. Neurons and astrocytes were identified histologically and immunohistochemically in neocortex, hippocampus, striatum, and cerebellum. However, most labeled cells in both gray and white matter appeared to be glia. Others have suggested that such Y-labeling represents fusion between host and donor cells, rather than true transdifferentiation. The possibilities of fusion and microchimerism were therefore examined using buccal epithelial cells as a model system. The female patients in this study had received either bone marrow or stem cell (CD34+ enriched) transplants from their brothers. Double labeling for X- and Y-chromosomes showed that Y-labeled buccal cells could not be explained by fusion. Genotyping studies of one patient, her brother, and her son ruled out the possibility of microchimerism. Whether, and under what circumstances, some form of bone marrow transplantation might provide adequate number of cells capable of replacing lost brain cells or enhancing their function will require additional studies.     

Differentiation of radial glia-like cells from embryonic stem cells

Radial glial cells play important roles in neural development. They provide support and guidance for neuronal migration and give rise to neurons and glia. In vitro, neurons, astrocytes, and oligodendrocytes can be generated from neural and embryonic stem cells, but the generation of radial glial cells from these stem cells has not yet been reported. Since the differentiation of radial glial cells is indispensable during brain development, we hypothesize that stem cells also generate radial glial cells during in vitro neural differentiation. To test this hypothesis, we utilized five different clones of mouse embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glia-like cells can be generated from ES/EC cell lines. These ES/EC cell-derived radial glia-like cells are similar in morphology to radial glial cells in vivo, i.e., they are bipolar with an unbranched long process and a short process. They also express several cytoskeletal markers, such as nestin, RC2, and/or GFAP, that are characteristics of radial glial cells in vivo. The processes of these in vitro generated radial glia-like cells are organized into parallel arrays that resemble the radial glial scaffolds in neocortical development. Since radial glia-like cells were observed in all five clones of ES/EC cells tested, we suggest that the differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system should facilitate the investigation of regulation of radial glial cell differentiation and its biological function.     

The tropism of embryoid body cells for glioma cells

It has been demonstrated that several types of adult stem cells have a common attribute of tropism for gliomas. In our study, we provided evidence that embryonic stem cell-derived embryoid body (EB) cells also exhibited a tropism for gliomas. Chemotaxis assays and organotypic hippocampal slice culture experiments showed that EB cells were attracted by the conditioned medium from C6 glioma cells and by C6 glioma cells deposited on the slice. Aggregate culture assays showed that EB cells could coaggregate with C6 glioma cells. Embryoid body cells injected intratumorally were found to distribute throughout the tumor mass. All data indicated that EB cells displayed a tropism for gliomas.