胶质瘤中肿瘤干细胞的分离、培养及鉴定

目的在胶质瘤中分离，培养及鉴定脑肿瘤干细胞，观察脑肿瘤干细胞的生长特性，建立脑肿瘤干细胞分离、培养及鉴定的方法，为脑肿瘤干细胞的进一步研究打下基础。方法术中取胶质瘤细胞，接种于含生长因子的无血清培养基中培养，使其中的脑肿瘤干细胞增殖，并进行有限稀释实验确定肿瘤比例及增值能力；利用细胞免疫荧光及组织免疫组化法检测脑肿瘤干细胞在细胞培养或组织切片中CD133和Nestin的表达。结果在胶质瘤中，只有大约1％的细胞能在无血清培养基中存活并悬浮生长，并增殖形成克隆性脑肿瘤干细胞球，传代后脑肿瘤干细胞仍保持很强的自我更新和增殖能力；体外培养中脑肿瘤干细胞表达神经干细胞的特异性标志物Nestin和CD133，在肿瘤组织切片中表达CD133阳性细胞。结论胶质瘤组织中存在极少数的脑肿瘤干细胞（大约为1％），并能在体外将其分离，培养；神经干细胞可能是脑肿瘤干细胞的起源细胞。     

人脑胶质母细胞瘤肿瘤干细胞的分离鉴定研究

目的 从人脑胶质母细胞瘤中分离出肿瘤干细胞.体外培养并鉴定其生物学特性.方法 获取5例人脑胶质母细胞瘤肿瘤细胞,通过无血清培养基和悬浮培养法,观察肿瘤干细胞克隆球的形成.利用免疫荧光技术检测原代肿瘤细胞和克隆球细胞中CD133阳性细胞的比例,分析克隆球细胞的抗原表达和多向分化能力.观察肿瘤干细胞子代分化细胞在无血清培养基中的逆向分化现象.结果 成功获取人脑胶质母细胞瘤肿瘤细胞,研究发现部分细胞具有克隆形成能力,在无血清培养基中呈悬浮球状生长,并可稳定增殖、传代.免疫荧光检测显示原代肿瘤细胞及克隆球细胞中均有CD133阳性细胞.克隆球细胞在含血清培养基中具有多向分化能力,其分化的子代细胞在无血清培养基中可逆向分化.结论 培养获得人脑胶质母细胞瘤肿瘤干细胞,证实其在体外具有自我更新、无限增殖、多向分化和逆向分化的生物学特性,为下一步研究奠定基础.     

CD133免疫磁珠分选脑肿瘤干细胞及其生物学特性

目的:从人脑肿瘤组织中分离、培养、纯化和鉴定脑肿瘤干细胞(BTSCs),探讨CD133免疫磁珠分选方法获取BTSCs的可行性及BTSCs的生物学特性.方法:留取人脑胶质母细胞瘤新鲜标本,分离获得脑肿瘤细胞.应用CD133免疫磁珠分选方法纯化BTSCs;流式细胞仪检测分选阳性率;神经球计数法分析CD133+/-亚群细胞神...     

胶质瘤干细胞的培养和分化研究

目的探讨人类胶质瘤干细胞的体外培养、传代和分化。方法采用机械方法从胶质瘤组织中分离细胞,应用N2培养基进行培养,碱性成纤维细胞生长因子和表皮生长因子刺激细胞扩增;应用免疫组织化学染色对培养的细胞及其分化的细胞进行鉴定。结果从胶质瘤组织当中成功培养出人类的胶质瘤干细胞,培养条件下呈悬浮状态生长,形成神经球,绝大多数的细胞表达波形蛋白和巢蛋白两种神经干细胞的标志物;这种细胞可分化为神经元和星型胶质细胞。结论体外的培养条件下,可从胶质瘤组织中培养出干细胞,能够分化为神经元和星型胶质细胞,为胶质瘤的深入研究莫定基础。     

CD133+ 胶质瘤干细胞化疗耐受机制分析

目的:探讨ABC超家族转运体蛋白在CD133 胶质瘤干细胞多药耐药性中的作用机制.方法:选取30例人脑胶质瘤标本,利用免疫磁珠法分选标本中的CD133 胶质瘤干细胞(悬浮细胞)和CD133-肿瘤细胞(黏附细胞),并进行分选细胞的体外扩增培养、传代与鉴定,应用免疫组化和RT-PCR法分别检测细胞中 MDR1和MRP1的蛋白及其活性表达情况.结果:3例胶质母细胞瘤来源的CD133 细胞能在含血清培养基中形成肿瘤干细胞球,并进行1～3次传代;MDR1与MRP1耐药蛋白在CD133 细胞中高度表达,阳性细胞比例范围分别为18%～67%和23%～73%;MDR1与MRP1在CD133 细胞中的表达活性分别为在CD133-细胞中的16.1倍和19.6倍;多药耐药蛋白的阳性细胞比例和其表达活性与肿瘤的恶性程度呈正相关,而表达阳性率与肿瘤的病理级别无关;MDR1与MRP1在CD133 细胞中的表达有明显的相关性.结论:神经上皮肿瘤中仅有小部分细胞亚型(CD133 胶质瘤干细胞)具有内在耐药性(天然耐药性),而ABC超家族转运体蛋白MDR1与MRP1在CD133 胶质瘤干细胞中的共同过度表达是胶质瘤多药耐药的主要原因之一;CD133 细胞是胶质瘤化疗的关键性治疗标靶.     

MRP-1、MDR1、GST-π mRNA在CD133~+肿瘤干细胞中的表达分析

目的分离鉴定CD133~+肿瘤干细胞,初步分析MRP-1、MDR1、GST-π在CD133~+胶质瘤干细胞多药耐药性中的作用。方法采用胶质瘤U+251细胞系、磁珠分离,培养CD133~+胶质瘤干细胞,RT-PCR技术分析MRP-1、MDR1、GST-π在CD133~+胶质瘤干细胞中的表达。结果培养的胶质母细胞瘤干细胞表达干细胞标记物Nestin,分化后细胞表达GFAP、β-tubulin;MRP-1、MDR1、GST-π在CD133~+胶质瘤干细胞中呈高表达,与CD133-胶质瘤干细胞比较差异有统计学意义(P0.05)。结论培养、鉴定胶质母细胞瘤肿瘤干细胞、MRP-1、M DR1、GST-π在CD133~+胶质瘤干细胞中呈高表达,为下一步研究奠定基础。     

胶质瘤细胞中干细胞分离的实验研究

目的研究应用无血清培养基悬浮法培养U-251胶质瘤细胞系的方法;并分离该细胞系中的脑肿瘤干细胞,鉴定其特异性标志物CD133+的表达并观察其生长和分化特征.方法应用培养大鼠神经干细胞的培养基和培养方法,在无血清培养基中采用悬浮法培养人U-251胶质瘤细胞系;再将分离获得的悬浮生长的脑肿瘤干细胞利用免疫荧光法检测脑肿瘤干细胞特异性标志物CD133+在脑肿瘤干细胞球中的表达.再将脑肿瘤干细胞接种于含血清培养基,观察其分化生长和分化特征.结果 U-251胶质瘤细胞系中有约(2.56±0.2)%的肿瘤细胞在无血清培养基中能够存活,增殖,形成自由漂浮的细胞球;其细胞表达CD133+神经干细胞的标志物,并可连续传代,若重新接种于含血清培养基中可重新贴壁分化,贴壁分化后细胞形态与直接在含血清培养基中培养的U-251胶质瘤细胞系无明显差别.结论用悬浮无血清培养法从U-251人胶质瘤细胞系中成功培养出脑肿瘤干细胞,其肿瘤干细胞能够产生为多种细胞形态的分化细胞,脑肿瘤干细胞的分离培养成功对脑肿瘤的基础和临床研究具有重要价值     

胶质瘤干细胞分离鉴定及其化疗敏感性研究

目的:探讨胶质瘤干细胞的分离、培养、鉴定及其与胶质瘤细胞化疗敏感性方面的差异.方法:将3株人胶质瘤细胞SHG-44、U251、U87MG分别接种于无血清培养基,从中分离出各自胶质瘤干细胞;扫描电镜观察胶质瘤干细胞的形态;RT-PCR法检测胶质瘤干细胞CD133、GFAP的表达;CCK8法检测化疗药物顺铂及长春新碱分别作用后胶质瘤细胞及其干细胞活性的变化.结果:从3株胶质瘤细胞中分离出的胶质瘤干细胞,可在体外增殖及传代,表达特异性标志物CD133,不表达GFAP.CCK8法检测出胶质瘤干细胞较胶质瘤细胞更为耐药(均P＜0.05).结论:胶质瘤干细胞较胶质瘤细胞化疗敏感性降低.     

神经上皮来源脑肿瘤中肿瘤干细胞的分离培养与鉴定

目的建立人神经上皮来源脑肿瘤中肿瘤干细胞的体外分离、培养体系,观察其分化现象,鉴定其特异性标志物,为脑肿瘤干细胞的进一步研究打下基础。方法将26例脑肿瘤手术中取出的脑肿瘤细胞,以2×10^5／ml的密度接种于含生长因子的无血清培养基培养,使其中的脑肿瘤干细胞增殖,并进行单克隆形成实验及溴脱氧尿苷掺入实验;然后将增殖形成的脑肿瘤干细胞球接种于无生长因子的含血清培养基诱导分化;利用免疫荧光和免疫组化法检测脑肿瘤干细胞在细胞培养和组织切片中CD133和巢蛋白的表达。结果各类肿瘤标本中仅有少数肿瘤细胞能够在无血清培养基中存活并悬浮生长,并增殖形成克隆性脑肿瘤干细胞球,传代后脑肿瘤干细胞保持很强的自我更新和增殖能力;在含血清培养基中脑肿瘤干细胞贴壁分化;体外培养中脑肿瘤干细胞表达神经干细胞的特异性标志物CD133和巢蛋白,在肿瘤组织切片中存在CD133阳性细胞,其阳性表达率为（21．0±6．2）％～（38．0±7．0）％。结论人神经上皮来源脑肿瘤组织中存在少量具有自我更新和增殖能力、表达CD133和巢蛋白的脑肿瘤干细胞,并能在体外将其分离、培养和诱导分化;脑肿瘤干细胞有可能成为脑肿瘤基础研究的新平台和临床治疗的重要靶细胞。     

立体定向建立裸鼠人脑多形性胶质细胞瘤模型及生长特性研究

目的:体外原代培养人脑多形性胶质母细胞瘤细胞,应用不同细胞数量接种裸鼠脑内建立颅内胶质瘤实验动物模型,观察其生长特性。方法:采用酶消化法原代培养4例人脑多形性胶质细胞瘤,分别取生长状态良好的胶质瘤细胞悬液20μL,按1.0×105个/10μL,1.0×106个/10μL,1.0×107个/10μL立体定向接种于裸鼠脑右侧尾状核区。在整体、组织和细胞水平观察并记录肿瘤生长情况,于成瘤后第5wk处死荷瘤鼠检测是否存在肿瘤远处转移,同时对肿瘤组织行HE和免疫组织化学染色检测胶质瘤的病理学特征。结果:细胞悬液接种可成功获得多形性胶质细胞瘤模型,成功率较高,但潜伏期延长,各种接种量的实验组成瘤率均为100%,未见颅外转移病灶,在组织病理学上接近人脑胶质瘤。结论:多形性胶质细胞瘤细胞接种裸鼠建立脑胶质瘤动物模型,成瘤率高,颅内生长稳定,肿瘤组织病理学及形态学特性与人脑胶质瘤相似,可作为临床胶质瘤基础研究的理想模型。     

脑肿瘤干细胞的分布与肿瘤微血管的相关性

目的 探讨脑肿瘤干细胞(BTSC)标记物CD133、Nestin和血管内皮细胞标记物CD31在脑胶质瘤中的表达.方法 (1)按照世界卫生组织(WHO)2000年的神经系统肿瘤分类分级标准所有标本分为Ⅱ级18例,Ⅲ级23例,Ⅳ级24例,采用免疫组织化学SP法检测CD133和Nestin在65例胶质瘤组织中的表达.(2)采用免疫荧光双染法检测CD133/CD31、Nestin/CD31和CD133/Nestin共表达情况.计算CD133~+肿瘤干细胞、CD133~+血管、Nestin~+肿瘤干细胞和Nestin~+血管所占的百分率,并进行统计学分析.结果 CD133~+肿瘤干细胞或Nestin~+肿瘤干细胞在各级别胶质瘤中均有表达,并且均可表达于血管内皮细胞.低级别组中,在CD133~+肿瘤干细胞或Nestin~+肿瘤干细胞分布的区域可见CD133~+血管或Nestin~+血管分布较少.在高级别组中,该区域则有丰富的CD133~+血管或Nestin~+血管分布,同时可见肿瘤干细胞包绕着血管生长.并且,CD133~+细胞或Nestin~+细胞的百分比与CD133~+血管(r=0.945,P<0.01)或Nestin~+血管(r=0.727,P<0.01)的表达呈正相关.免疫荧光双染可见,CD133~+细胞或Nestin~+细胞均聚集于CD31~+血管周围,并且,CD133~+/CD31~+、Nestin~+/CD31~+细胞共表达于血管内皮细胞.结论 在脑胶质瘤组织中,血管内皮细胞可表达肿瘤干细胞的标记物,因此认为,肿瘤内微血管可能来源于肿瘤干细胞的分化,并且肿瘤干细胞聚集于微血管周围生长呈巢状分布.随着肿瘤病理级别的升高,CD133~+肿瘤干细胞或Nestin~+肿瘤干细胞的百分比与CD133~+血管或Nestin~+血管的表达呈正相关.     

脑肿瘤干细胞与神经上皮肿瘤病理级别的相关性

目的：探讨脑肿瘤干细胞（brain tumor stem cells，BTSCs）的分离、培养及鉴定的方法，研究BTSCs比例与人脑神经上皮肿瘤病理级别之间的相关性。方法：将人神经上皮肿瘤细胞接种于含生长因子的无血清培养基中，使其中的脑肿瘤干细胞增殖和传代，利用细胞免疫荧光及免疫组化法检测干细胞的特异性标记物Nestin和CD133的表达；采用免疫组化（SABC法）研究46例脑肿瘤及5例正常脑组织CD133的表达，并与肿瘤病理分级进行相关性分析。结果：可以从人神经上皮肿瘤病例中分离培养出BTSCs；BTSCs可以在体外增殖并传代，并且表达干细胞的特异性标记物Nestin和CD133；CD133在正常的脑组织中未见表达，在各级神经上皮肿瘤中均有表达，各级别肿瘤中的阳性细胞比例差异有显著性（P〈0．01），CD133阳性细胞比例与肿瘤的恶性程度呈正相关（P〈0．01）。结论：各种病理类型神经上皮肿瘤组织中均存在干细胞特征的细胞，CD133的表达与肿瘤级别呈正相关。     

Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells

Background  Recent studies have suggested that cancer stem cells cause tumor recurrence based on their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma cells is also implicated in the failure of current therapies, it is not clear whether cancer stem cells are involved in invasiveness. This study aimed to assess invasive ability of glioma stem cells (GSCs) derived from C6 glioma cell line and the distribution patterns of GSCs in Sprague-Dawley (SD) rat brain tumor. 

Methods  Serum-free medium culture and magnetic isolation were used to gain purely CD133⁺ GSCs. The invasive ability of CD133⁺ and CD133^– C6 cells were determined using matrigel invasion assay. Immunohistochemical staining for stem cell markers and luxol fast blue staining for white matter tracts were performed to show the distribution patterns of GSCs in brain tumor of rats and the relationship among GSCs, vessels, and white matter tracts. The results of matrigel invasion assay were estimated using the Student’s t test and the analysis of Western blotting was performed using the one-way analysis of variance (ANOVA) test.

Results  CD133⁺ GSCs (number: 85.3±4.0) were significantly more invasive in vitro than matched CD133^– cells (number: 25.9±3.1) (t=14.5, P <0.005). GSCs invaded into the brain diffusely and located in perivascular niche of tumor-brain interface or resided within perivascular niche next to white fiber tracts. The polarity of glioma cells containing GSCs was parallel to the white matter tracts.

Conclusions  Our data suggest that CD133⁺ GSCs exhibit more aggressive invasion in vitro and GSCs in vivo probably disseminate along the long axis of blood vessels and transit through the white matter tracts. The therapies targeting GSCs invasion combined with traditional glioblastoma multiforme therapeutic paradigms might be a new approach for avoiding malignant glioma recurrence.

     

多药耐药基因MGMT在脑胶质瘤干细胞中表达的实验研究

目的:探讨甲基鸟嘌呤DNA甲基转移酶(Methylguanine-dna methyltransferase,MGMT)在脑胶质瘤细胞和胶质瘤干细胞(Brain glioma stem cells,BGSC)中的表达情况.方法:由手术切除的多形性胶质母细胞瘤中分离培养胶质瘤细胞和BGSC;应用免疫细胞化学染色和流式细胞...     

Rac1+ cells distributed in accordance with CD 133+ cells in glioblastomas and the elevated invasiveness of CD 133+ glioma cells with higher Rac1 activity

Background  Recent studies have suggested that cancer stem cells are one of the major causes for tumor recurrence due to their resistance to radiotherapy and chemotherapy. Although the highly invasive nature of glioblastoma (GBM) cells is also implicated in the failure of current therapies, it is not clear how glioma stem cells (GSCs) are involved in invasiveness. Rac1 activity is necessary for inducing reorganization of actin cytoskeleton and cell movement. In this study, we aimed to investigate the distribution characteristics of CD133+ cells and Rac1+ cells in GBM as well as Rac1 activity in CD133+ GBM cells, and analyze the migration and invasion potential of these cells.

Methods  A series of 21 patients with GBM were admitted consecutively and received tumor resection in Tianjin Medical University General Hospital during the first half of the year 2011. Tissue specimens were collected both from the peripheral and the central parts for each tumor under magnetic resonance imaging (MRI) navigation guidance. Immunohistochemical staining was used to detect the CD133+ cells and Rac1+ cells distribution in GBM specimens. Double-labeling immunofluorescence was further used to analyze CD133 and Rac1 co-expression and the relationship between CD133+ cells distribution and Rac1 expression. Serum-free medium culture and magnetic sorting were used to isolate CD133+ cells from U87 cell line. Rac1 activation assay was conducted to assess the activation of Rac1 in CD133+ and CD133- U87 cells. The migration and invasive ability of CD133+ and CD133- U87 cells were determined by cell migration and invasion assays in vitro. Student’s t-test and one-way analysis of variance (ANOVA) test were used to determine statistical significance in this study.

Results  In the central parts of GBMs, CD133+ cells were found to cluster around necrosis and occasionally cluster around the vessels under the microscope by immunohistological staining. In the peripheral parts of the tumors, CD133+ cells were lined up along the basement membrane of the vessels and myelinated nerve fibers. Rac1 expression was high and diffused in the central parts of the GBMs, and the Rac1+ cells were distributed basically in accordance with CD133+ cells both in the central and peripheral parts of GBMs. In double-labeling immunofluorescence, Rac1 was expressed in (83.14±4.23)% of CD133+ cells, and CD133 and Rac1 co-expressed cells were located around the vessels in GBMs. Significantly higher amounts of Rac1-GTP were expressed in the CD133+ cells (0.378±0.007), compared to CD133- cells (0.195±0.004) (t=27.81; P <0.05). CD133+ cells had stronger ability to migrate (74.34±2.40 vs. 38.72±2.60, t=42.71, P <0.005) and invade (52.00±2.28 vs. 31.26±1.82, t=30.76, P <0.005), compared to their counterpart CD133- cells in transwell cell migration/invasion assay.

Conclusions  These data suggest that CD133+ GBM cells highly express Rac1 and have greater potential to migrate and invade through activated Rac1-GTP. The accordance of distribution between Rac1+ cells and CD133+ cells in GBMs implies that Rac1 might be an inhibited target to prevent invasion and migration and to avoid malignant glioma recurrence.

     

CD133+细胞在局部晚期结肠癌中的异常表达

目的 探讨CD133+肿瘤细胞对结肠癌患者预后的影响。方法 收集有完整随访资料的104例IIIB期(T3-4N1M0)结肠癌根治术后石蜡包埋组织标本及临床资料,采用免疫组化方法检测癌组织及邻近癌旁相对正常组织中CD133抗原的表达情况,分析其与患者临床特征和预后的关系。结果 肿瘤组织中的CD133+肿瘤细胞比较少见而且分布不均匀。CD133染色见于结肠癌细胞膜的腺腔面及基底,在癌巢"出芽"处及有腺管结构的低分化腺癌也可见CD133染色。生存分析显示CD133及T分期都是IIIB结肠癌的独立预后因素。肿瘤组织中CD133+细胞＜5%的患者5年生存率较高（77.4%）,≥ 5％则为45.2%。CD133的表达与其他临床病理参数均无相关性。结论 CD133+肿瘤细胞是IIIB期结肠癌患者5年生存率的独立预后因素, CD133+肿瘤细胞促进了IIIB期结肠癌的进展。     

免疫磁珠法分离纯化人肾癌CD133~+细胞实验研究

目的:本研究通过分析CD133+细胞在肾细胞癌患者肿瘤组织的表达情况,寻找肾癌中CD133+细胞分离纯化方法,探寻CD133+细胞与肾癌发生中的作用。方法:①应用胎盼蓝拒染试验检测细胞活性。②应用免疫磁珠法分离纯化肿瘤组织中CD133+细胞。③应用流式细胞仪检测肾透明细胞癌标本中CD133+细胞表达情况。结果:30例肾透明细胞癌中CD133+比为(13.08±1.39)%,与病理分级、临床分期无关。免疫磁珠法分离纯化CD133+纯度为(75±2.4)%,细胞活性不受影响。结论:免疫磁珠技术纯化肾癌CD133+细胞成熟有效,CD133+细胞可能是肾癌干细胞。     

HuH7细胞系CD13~+CD133~+HCC细胞分选及CSCs特性分析

目的探讨CD13、CD133双荧光标志流式细胞术(fluorescence activated cell sorter,FACS)分选人肝癌HuH7细胞系CD13+CD133+HCC细胞方法及癌干细胞(cancer stem cells,CSCs)特征。方法 CD13、CD133双标志,采用FACS从人肝癌HuH7细胞系分选出CD13+CD133+HCC、CD13+CD133-HCC、CD13-CD133+HCC、CD13-CD133-HCC等4种细胞亚群;通过CD13+CD133+HCC细胞生长曲线,细胞周期,形成肿瘤球和裸鼠体内成瘤能力,及对5-FU、吡柔比星化疗药物的敏感性研究,分析CD13+CD133+HCC细胞是否具有CSCs生物学特征。结果人肝癌HuH7细胞系CD13+CD133+HCC细胞占23.8%,3.1%分选为CD13+CD133+HCC细胞,78.45%的CD13+CD133+HCC细胞处于G0/G1期;CD13+CD133+HCC细胞增殖明显快于其他3个细胞亚群;在裸鼠103个CD13+CD133+HCC细胞就可以成瘤,而1.0×105个CD13-CD133-HCC只有2只成瘤(2/5),干细胞培养CD13+CD133+HCC细胞8～15 d形成肿瘤球;CD13+CD133+HCC细胞对5-FU和吡柔比星具有抵抗特性,而其他3个亚群较易于杀灭。结论 CD13、CD133双标志FACS从HuH7分选的CD13+CD133+HCC细胞具有CSCs特征,可能成为HCC治疗靶细胞。