Rac1在脑胶质瘤干细胞迁移和侵袭中的作用

目的 通过特异性的Racl活性抑制剂下调胶质瘤干细胞(GSCs)中Rac1的活性,探讨Rac1在GSCs迁移和侵袭中的作用. 方法 将U251细胞置于无血清DMEM/F12干细胞培养基中培养,免疫磁珠分选法分离GSCs,免疫荧光染色检测CD133鉴定GSCs; Rac1活性实验检测CD133+与CD133-细胞活性,Transwell细胞迁移和侵袭实验评价CD133+与CD133-细胞的迁移和侵袭能力;加入Rac1活性抑制剂NSC23766处理GSCs,活性实验检测细胞Racl活性,Western blotting检测hMena和基质金属蛋白酶9(MMP-9)蛋白的表达;Transwell细胞迁移实验和侵袭实验评价细胞迁移和侵袭能力的改变. 结果 成功培养出悬浮生长的细胞球,可以连续传代并持续表达神经干细胞标志物CD133;CD133+细胞Rac1-三磷酸鸟苷(GTP)表达水平、细胞迁移和侵袭的能力显著高于CD133-细胞,差异有统计学意义(P＜0.05);与对照组比较,NSC23766处理组GSCs的Rac 1-GTP、hMena和MMP-9蛋白表达水平明显降低,迁移和侵袭能力明显减弱,差异有统计学意义(P＜0.05). 结论 GSCs相对于肿瘤中的其他细胞具有更强的迁移和侵袭能力,Rac1对脑GSCs的迁移和侵袭具有调控作用,抑制Rac1活化可能成为治疗恶性胶质瘤的新策略.     

BMP受体在胶质母细胞瘤起始细胞早期分化中的表达

目的在分离鉴定胶质母细胞瘤U87细胞中的胶质瘤起始细胞(GTICs)的基础上,检测在GTICs细胞中骨形态发生蛋白(BMP)受体1A、B的表达,探讨BMP受体1A、B对GTICs所起的生物学作用。方法利用流式细胞术检测U87细胞中CD133和巢蛋白阳性细胞,细胞免疫荧光和蛋白印迹法检测GTICs和普通细胞中BMP受体1A、B的表达。结果 CD133和巢蛋白阳性表达的细胞在瘤U87细胞中所占的比例分别0.92%和5.72%;GTICs可诱导分化为表达β-微导管蛋白和胶质纤维酸性蛋白的细胞;U87细胞的GTICs中BMP受体1A表达明显增高,而经诱导分化培养后的BMP受体1A的表达又明显降低,BMP受体1B表达变化不明显。结论 BMPs信号通路中BMP受体对调控GTICs的增殖分化可能起着重要作用。     

siRNA靶向沉默组织蛋白酶D对胶质瘤细胞生物学行为的影响

目的 探讨小分子干扰RNA(siRNA)靶向沉默胶质瘤细胞系U87中组织蛋白酶D(CD)对胶质瘤细胞生物学行为的影响. 方法 采用脂质体介导的CD siRNA、scramble siRNA、空白对照siRNA转染体外常规培养的胶质瘤细胞株U87,RT-PCR、Western blotting分别检测转染后U87细胞CD mRNA和蛋白的表达;MTT法检测转染CD siRNA、scramble siRNA 1～5 d后U87细胞的增殖;细胞侵袭实验检测转染CD siRNA、scramble siRNA 48 h后细胞侵袭能力的变化. 结果 转染48 h后CD siRNA组细胞CD mRNA、蛋白的表达明显低于转染scramble siRNA和空白对照siRNA组,差异有统计学意义(P＜0.05);转染后2、3、4、5d转染CD siRNA组U87细胞吸光度(A)值低于转染scramble siRNA组,差异有统计学意义(P＜0.05);细胞侵袭实验结果表明转染CDsiRNA组滤膜细胞数低于转染scramble siRNA组,差异有统计学意义(P＜0.05). 结论 CD可能是具有广泛应用前景的胶质瘤分子生物治疗的靶点.     

白藜芦醇对脑胶质瘤U87细胞及干细胞凋亡诱导作用

目的比较研究白黎芦醇(Res)对人脑胶质瘤U87细胞及其胶质瘤干细胞(GSC)的作用。方法膜联蛋白Ⅴ(AnnexinⅤ)和碘化丙啶(PI)染色法测定细胞凋亡;CD133标记、CD133与AnnexinⅤ/PI共标记检测GSC相对含量及GSC的凋亡;实时荧光定量逆转录-聚合酶链式反应(RT-PCR)检测mdr1基因mRNA表达,流式细胞术(FCM)测定P-糖蛋白(P-gp)的表达;集落形成法(CFU)检测细胞的自我更新和增殖能力。结果不同浓度的Res显著诱导U87细胞凋亡,抑制U87细胞集落的形成。40～160μmol/L Res作用后U87细胞群体中CD133+GSC相对含量显著增高、GSC凋亡细胞数量增加,但GSC对Res的敏感性低于U87群体细胞。Res诱导后U87细胞群体中高表达mdr1/P-gp的细胞显著增高,并与GSC含量的增高基本相一致。结论白黎芦醇可诱导脑恶性胶质瘤U87群体细胞及其干细胞凋亡。     

Nanog基因在脑肿瘤干细胞中的表达及意义

目的 探讨Nanog基因在脑肿瘤干细胞(BTSCs)的表达以及生物学意义.方法 无血清悬浮培养法从U87胶质瘤细胞株分离培养BTSCs.免疫细胞化学染色和RT-PCR方法检测U87肿瘤细胞及其BTSCs中Nanog的表达,双重免疫细胞化学染色法检测Nanog/CD133共表达情况.结果 在U87胶质瘤细胞株中成功分离培养出BTSCs,Nanog蛋白在U87肿瘤细胞和BTSCs中的阳性率分别为(64.1±18.2)%和(97.2±1.8)%,且Nanog+/CD133+细胞共表达于部分细胞中;Nanog mRNA相对含量在U87肿瘤细胞和BTSCs中分别为0.3851±0.0771和0.6032±0.1223,差异有统计学意义(P＜0.05).结论 U87细胞株中存在BTSCs.Nanog在U87细胞株中高表达,在BTSCs中表达水平高于U87肿瘤细胞且Nanog与CD133存在共表达,表明Nanog与BTSCs关系密切.

Abstract：

Objective To detect the expression of Nanog in glioma cell line U87 and the relationship with BTSCs.Methods BTSCs were isolated from glioma cell line U87 and cultured in simplified serum-free neural stem cell medium by nanosphere suspension culture method spheres,and purified continuously through the monoclonal formation experiment.The immunofluorescence staining of cells was employed to identify the BTSCs and differentiated cells.The expression of Nanog mRNA was examined by RT- PCR and Nanog protein was detected by immunocytochemistry in U87 and BTSCs respectively.Double immunocytochemistry staining was used to detect the co- expressions of Nanog/CD133.Methods BTSCs were isolated ,cultured and purified successfully from glioma cell line U87.Both Nanog mRNA and protein were expressed in U87 and BTSCs.The expression rate of immunopositive cells was (64.1 ±18.2)% in U87 and (97.2±1.8)% in BTSCs respectively(t =5.719,P =0.000).The Nanog+/CD133+ cells could be found co-expressed in U87 and BTSCs by using double immunocytochemistry s taining.The relative level of Nanog expression was 0.3851 ±0.0771 in U87 and0.6032±0.1223 in BTSCs(t =4.770,P =0.000).Conclusion BTSCs exist in glioma cell line U87 in vitro.The levels of Nanog expression in BTSCs were higher than that in U87.Nanog expression was associated with the genesis and differentiation state of glioma.The overexpression of Nanog and co- expression of Nanog/CD133 showed that it might contribute to the existence of BTSCs,which lay a foundation to the further explore its role in biological behaviour of glioma.Expression of Nanog in U87 indicated the close relationship between glioma and stem cell,and suggested Nanog may play a critical role in the development of glioma.     

Oct-4在激素非依赖性前列腺癌干细胞中的表达及意义

背景：干细胞特异性基因Oct-4在维持干细胞功能和调节干细胞的分化过程中起着关键性作用,了解前列腺癌干细胞中Oct-4的表达,对前列腺癌的预后、复发和耐药的评估具有重要意义。 目的：观察Oct-4在激素非依赖性前列腺癌干细胞中的表达。 设计、时间及地点：观察性实验,于2008-02/10在南昌大学第一附属医院中心实验室完成。 材料：取12例前列腺癌患者手术后组织标本,其中6例在手术前没有经过化疗和激素治疗,另外6例患者经过激素治疗出现抵抗而手术。 方法：制备前列腺癌组织单细胞悬液,流式细胞仪分选未经治疗前列腺癌和治疗后出现耐药的前列腺癌干细胞,RT-PCR和Western blot检测激素非依赖性前列腺癌Oct-4基因和蛋白的表达。 主要观察指标：激素依赖和非依赖性前列腺癌CD133+/CD44+以及CD133-/CD44-细胞的比例;Oct-4基因和蛋白在激素非依赖性前列腺癌CD133+/CD44+、CD133+/CD44-、CD133-/CD44+、CD133-/CD44-细胞的表达。 结果：流式细胞仪检测显示未经过治疗的前列腺癌CD133+/CD44+细胞占(0.83±0.21)%,而CD133-/CD44-细胞多达(95.62±1.35)%;治疗出现激素抵抗的前列腺癌CD133+/CD44+细胞为(54.62±0.86)%,而CD133-/CD44-细胞为(9.56±0.47)%;与其他亚群细胞相比,激素非依赖性前列腺癌CD133-/CD44-细胞Oct-4基因和蛋白显著降低(P < 0.01);CD133+/CD44+细胞Oct-4基因和蛋白表达显著升高(P < 0.01);而CD133+/CD44-和CD133-/CD44+细胞之间Oct-4基因和蛋白表达无显著差异(P > 0.05)。 结论：Oct-4在激素非依赖性前列腺癌干细胞中高表达,可能与前列腺癌的预后和耐药有关。     

ADAM12基因表达沉默对CD133阳性胶质瘤细胞自我更新能力的影响

目的研究解整合素-金属蛋白酶12(a disintegrin and metalloprotease 12,ADAM12)基因表达沉默抑制CD133阳性(CD133+)胶质瘤细胞的自我更新能力。方法采用sh RNA重组慢病毒转染技术沉默胶质瘤U87细胞系ADAM12基因表达分为ADAM12基因表达干扰序列组(sh RNA-ADAM12)、阴性对照组(sh RNA-NC)及空白对照(sh RNA-C)组。通过Western blotting以及Real-time PCR验证各组细胞ADAM12表达情况;采用悬浮培养得到胶质瘤细胞球以富集CD133+的胶质瘤细胞;并通过免疫荧光染色技术检测ADAM12与CD133在细胞球与贴壁细胞的表达情况;通过神经肿瘤球形成实验检测三组细胞的自我更新能力;利用Western blotting分别检测三组细胞成球后未分化或分化相关蛋白CD133、GFAP及TUBB3以及Notch通路靶基因Hes1的蛋白表达情况。结果慢病毒转染技术可显著下调U87胶质瘤细胞ADAM12的m RNA及蛋白的表达量,sh RNA-ADAM12组与sh RNA-NC组较sh RNA-C组m RNA的相对表达量为0.22±0.03与0.98±0.06(F=425.37,P0.01);三组ADAM12蛋白的相对表达量分别为28.72%±2.36%、69.21%±3.92%及69.04%±3.57%(F=145.42,P0.01);免疫荧光染色显示细胞球中ADAM12与CD133表达量明显高于普通细胞;神经肿瘤球形成实验结果显示,三组成球数分别为45.5±2.3、104.2±5.8以及109.6±6.2,与sh RNA-NC组及sh RNA-C组相比,sh RNA-ADAM12组的成球能力明显降低,差异具有统计学意义(F=147.03,P0.01)。sh RNA-ADAM12组与sh RNA-C组相比,GFAP与TUBB3蛋白表达量分别上调约166%与146%,CD133与HES1的蛋白表达量分别下调了54%与50%,差异均具有统计学意义(P0.01)。结论 ADAM12基因表达沉默可能通过抑制Notch通路活性降低CD133+胶质瘤细胞的自我更新能力。     

脑胶质瘤干细胞鉴定与增殖及耐药特性的实验研究

目的 从胶质瘤组织中分离和培养出肿瘤干细胞,并初步探讨其生长特性. 方法 收集脑胶质瘤手术标本并获取细胞,用含有表皮生长因子(EGF)、白血病细胞抑制因子(LIE)和碱性成纤维生长因子(bFGF)的无血清培养液原代培养,再经免疫磁珠分离得到CD133+细胞.并用免疫细胞化学技术检测CD133、NSE和GFAP在细胞中的表达以鉴定CD133+细胞,比较不同恶性级别胶质瘤组织的CD133+细胞生长情况,并用CCK8法比较CD133+和CD133-细胞对替尼泊苷(VM-26)的耐药性. 结果 从胶质瘤组织中成功分选获得CD133+细胞,这些细胞能自我更新,增殖,并分化成NSE+和GFAP+的细胞.恶性度高的胶质瘤组织中CD133+细胞生长速度明显比低级别中的CD133+细胞快,且CD133+细胞在含有VM-26培养基中的存活细胞数显著多于CD133-细胞(P＜0.05). 结论 胶质瘤组织中存在肿瘤干细胞,这类细胞具有很强的耐药性,高恶性度胶质瘤组织中的CD133+细胞具有更强的增殖能力.     

促自噬剂AZD8055对U87起始细胞Notch通路相关蛋白的表达和成球能力的影响

目的研究促自噬剂AZD8055对U87细胞株起始细胞Notch通路相关蛋白的表达和成球能力的影响。方法应用免疫磁珠法分选获得U87细胞株CD133阳性的胶质瘤起始细胞（GICs）。采用免疫荧光染色技术鉴定GICs细胞球干性标志物CDl33和Nestin,以及自噬相关蛋白LC3B和p62的表达。以CCK8检测AZD8055对U87起始细胞存活率的影响。采用Western blot检测AZD8055不同浓度处理组Notch1、Notch胞内域、Hes1、p62、LC3B蛋白的表达。通过单细胞成球实验观察AZD8055对GICs成球能力的影响。结果免疫荧光染色结果显示,GICs细胞球中CD133和Nestin呈阳性表达。CCK8检测结果提示,随着AZD8055浓度的增高,U87起始细胞的存活率逐渐降低。Western blot结果提示,与溶媒对照组相比,随着AZD8055浓度的升高,药物处理组的自噬水平增高,而Notchl通路相关蛋白的表达降低,且各浓度组之间的蛋白表达差异均有统计学意义（均P〈0．05）。免疫荧光染色结果提示,经AZD8055处理后U87起始细胞的LC3B表达明显升高,p62表达降低。单细胞成球实验显示,随着AZD8055药物浓度的增高,各组成球数目和细胞球体积递减,且差异有统计学意义（均P〈0．05）,说明U87GICs的成球能力降低。结论AZD8055通过诱发自噬抑制了Notch通路相关蛋白的表达,并且AZD8055能够抑制U87GICs的成球能力。     

Celecoxib enhances radiosensitivity in medulloblastoma-derived CD133-positive cells

Objects

Cyclooxygenase-2 (COX-2), the enzyme that converts arachidonic acid to prostaglandins, is overexpressed in a variety of tumors, including medulloblastoma (MB). CD133, a transmembrane glycoprotein, has been suggested as a marker for cancer stem cells in brain tumors. The aim of the present study was to investigate the role of celecoxib, a selective COX-2 inhibitor, in enhancing the effects of ionizing radiotherapy (IR) on medulloblastoma-derived CD133-positive cells (MB-CD133⁺).

Materials and methods

MB-CD133⁺ were isolated from two medulloblastoma cell lines (Daoy and UW228). Then, they were treated with celecoxib in different concentrations, and cell viability was assessed. The assays of cell survival, soft agar, radiosensitivity, colony formation, and apoptotic activity in MB-CD133⁺ treated with celecoxib alone, radiation alone, or celecoxib combined with radiation were further evaluated.

Results

MB-CD133⁺ showed the self-renew ability to form sphere bodies in vitro and regenerate tumors in vivo. The levels of COX-2 mRNA and protein in MB-CD133⁺ were significantly higher than those in MB-CD133^?. The treatment of 30 μM celecoxib could effectively inhibit the abilities of cell proliferation and colony formation and increase IR-induced apoptosis in treated MB-CD133⁺. Furthermore, in vivo study demonstrated that celecoxib significantly enhanced radiosensitivity in MB-CD133⁺-transplanted grafts. Notably, xenotransplantation analysis demonstrated that the treatment of celecoxib could further suppress the expressions of angiogenic and stemnness-related genes in treated MB-CD133⁺ grafts of SCID mice.

Conclusions

Celecoxib presents the potential of radiosensitizing effect in MB-derived cancer stem cells. Therefore, it should be warranted in future trials to enhance the radiotherapeutic effects in MB patients.     

CD133鉴定胶质瘤干细胞血管周围壁龛的实验研究

目的 检测脑肿瘤干细胞(BTSC)标记物CD133、巢蛋白(Nestin)和增殖细胞核抗原(PCNA)在74例脑胶质瘤标本中的表达,探讨肿瘤干细胞生存的微环境一壁龛的组成、形态及其在脑肿瘤组织中的分布. 方法 选取安徽医科大学附属省立医院神经外科自2007年1月至2008年10月间手术切除的74例胶质瘤标本,按照WH02000年的神经系统肿瘤分类分级标准分为Ⅱ级22例(低级别组)、Ⅲ级27例和Ⅳ级25例(高级别组),采用免疫组织化学染色和免疫荧光双标法分别检测标本中CD133的表达及其与Nestin、PCNA的共表达情况.计算并比较不同级别胶质瘤组织CD133+细胞、CD133+血管和CD133+壁龛所占的百分比.并对CD133+血管和CD133+壁龛的表达进行相关性分析. 结果 CD133+细胞聚集于壁龛内生长,低级别组胶质瘤中CD133+壁龛阳性率较低.壁龛内增殖细胞较少,与相邻肇龛之间界限清晰,周围CD133+血管分布较少.高级别组胶质瘤中CD133+壁龛阳性率高,壁龛之间无明显界限,壁龛内细胞增殖活跃,周围可见丰富的CD133+血管分布:壁龛中除CD133+/Nestin+BTSC外,可见CD133+/Nestin-细胞、CD133/PCNA+细胞等亚群细胞;不同级别胶质瘤CD133+细胞、CD133+血管、CD133+壁龛百分比不同,且肿瘤级别越高,三者表达越高,差异有统计学意义(P<0.05).CD133+壁龛与CD133+血管的表达呈正相关(r=0.425,P=0.000). 结论 在脑胶质瘤组织中存在着由CD133+/Nestin+BTSC和一些亚群细胞组成的壁龛结构,CD133+血管对于壁龛结构的维持起着非常重要的作用.     

CD133+ glioblastoma stem-like cells induce vascular mimicry in vivo

Glioblastoma is one of the most angiogenic malignancy, the neoplastic vessels of which are likely to arise by angiogenesis and vasculogenesis. An alternative mechanism of tumor vasculature is described, termed vasculogenic mimicry, by which highly aggressive tumor cells can form vessel-like structures themselves, by virtue of their high cellular plasticity. Evidence suggests that cancer stem cells acquire a multi-potent plastic phenotype and show vasculogenic potential. In this study, we report that glioblastoma stem-like cells (GSCs) can form vasculogenic mimicry in tumor xenografts and express pro-vascular molecules. We isolated GSCs from resected human glioblastoma tissues and demonstrated their stemness, differentiation, and in vivo tumor-initiating potential. Through a limiting dilution assay, CD133+ (CD133(+)-GSC) and CD133- (CD133(-)-GSC) subpopulation of GSCs were obtained. Orthotopic xenotransplantation study revealed that these two subpopulations of GSCs shared similar efficacy in tumor formation but showed distinct intratumor vasculature. In comparison with CD133(-)-GSC, a highly vascularized anaplastic tumor, mimicking vasculogenic mimicry, was found in CD133(+)-GSC-derived tumor xenografts. Subsets of CD133(+)-GSC but not CD133(-)-GSC were capable of vascular smooth muscle-like cell differentiation, in vitro and in vivo. In tumor xenografts, endothelium-associated CD31 gene was detected in implanted CD133(-)-GSC and exclusively dispersed within the tumor tissues. Although the detailed action mechanisms required further investigation, this study demonstrated the vasculogenic capacity of brain GSCs and their cellular plasticity. The results of expression of pro-vascular molecules and differentiation of vascular-like cells suggest that GSCs may contribute to form vessel-like structures and provide a blood supply for glioblastoma cells.     

大肠癌患者外周血细胞CD133的表达***

背景：近期有报道称在肿瘤患者外周血中检测出了高于正常值的CD133阳性细胞。 目的：观察CD133 mRNA和蛋白在大肠癌患者外周血中的表达并探讨其临床意义。 方法：应用流式细胞技术与RT-PCR反应检测29例大肠癌患者和20例正常对照外周血中CD133 抗原与mRNA的表达。 结果与结论：发生转移的大肠癌患者外周血中CD133阳性细胞比例显著高于正常对照及无转移的大肠癌患者(P < 0.01)。正常健康人群和无转移大肠癌患者外周血中CD133mRNA表达为阴性,发生转移的部分大肠癌患者外周血中CD133mRNA表达阳性,阳性率为35.7%(5/14),在超过38个以上循环时表达为弱阳性。结果表明,无转移大肠癌患者外周血中CD133 mRNA和蛋白表达与正常对照无区别,发生转移的大肠癌患者外周血中CD133 mRNA和蛋白高于正常对照和未发生转移者。     

Presence of pluripotent CD133+ cells correlates with malignancy of gliomas

BackgroundPresence of CD133⁺ cancer stem cells has been demonstrated within glioblastoma multiforme (GBM), the most malignant phenotype of gliomas (WHO grade IV). Since GBM frequently develops from low grade gliomas (WHO grade II) we assessed a possible qualitative or quantitative correlation of CD133⁺ cells and glioma grade to get new insights in gliomagenesis.ResultsThe amount of CD133⁺ cells within the bulk tumor mass, analyzed by immunostaining and Western blotting, showed a clear quantitative correlation with glioma grade (WHO° II, III and IV). Most of CD133⁺ cells were arranged in clusters frequently associated to tumor vessels. Protein analysis revealed high cellular coexpression of CD133 with Musashi-I but not CD34 indicating a neural, i.e. local origin of these cells. In vitro, no differences in stem cell properties concerning self-renewal and multi-lineage differentiation have been found for CD133⁺ cells isolated from gliomas of different grades.ConclusionsThese findings indicate a solely quantitative correlation of glioma grade with the presence of neural CD133⁺ cells within tumors supporting the concept of a CD133⁺ stem cell dependent gliomagenesis.     

无血清悬浮培养肾癌干细胞表面标志CD133及端粒酶活性的表达

背景：有研究表明端粒酶活性抑制剂不仅能抑制或杀死肾癌细胞,而且对决定肾癌发生发展的干细胞也有作用。 目的：观察无血清悬浮培养的肾癌干细胞表面标志CD133及端粒酶活性的表达情况,并与含血清培养的肾癌细胞作比较。 设计、时间及地点：细胞学体外观察,于2008-06/2009-02在江苏大学完成。 材料：手术切除肾癌周围新鲜的正常肾组织标本由江苏大学附属医院提供,肾癌干细胞株OS-RC-2由上海中科院细胞库提供。 方法：取处于对数生长期的肾癌干细胞OS-RC-2,胰酶消化后离心弃上清,悬浮于含EGF、bFGF的DMEM/F12无血清培养基中,调整细胞浓度为2×108 L-1进行接种,在37 ℃、体积分数为5%的CO2培养箱中悬浮培养。以含血清培养的肾癌细胞及正常肾组织细胞作为对照。 主要观察指标：倒置显微镜下观察细胞生长情况,流式细胞仪检测肾癌干细胞CD133及CD34的表达,采用TRAP实时定量检测肾癌干细胞端粒酶活性。 结果：肾癌干细胞接种于无血清培养基后,细胞呈圆形悬浮于培养液中;2 d后有细胞球生成,每个细胞球含3~8个细胞,折光性较强;7 d后细胞球明显增多,体积增大,为规则的圆形或卵圆形。无血清悬浮培养的肾癌干细胞球CD133+CD34-率明显高于含血清培养的肾癌细胞CD133+CD34-率,正常肾组织细胞未测出有CD133及CD34的表达,3者间比较差异有显著性意义(F=328.25,P < 0.05)。肾癌干细胞球和肾癌细胞端粒酶活性均高于正常肾组织细胞(F=278.74,P < 0.05),而前2者间端粒酶活性无明显差异。 结论：与含血清培养的肾癌细胞相比,无血清悬浮培养的肾癌干细胞表面标志CD133呈明显高表达,二者端粒酶活性均高于正常肾组织。     

A novel fixative for immunofluorescence staining of CD133-positive glioblastoma stem cells

Isolation of glioblastoma stem cells requires incubation of tumor cells in a neural stem cell media. Neurospheres containing these glioblastoma stem cells are formed after approximately a five-day period. These cells can then be analyzed for the presence of stem cell markers. Immunofluorescence staining for these markers can serve as a valuable tool for analyzing the intact neurosphere directly in stem cell media. Here we present the use of a novel fixative (1,4-benzoquinone) for immunoflourescence staining of neurospheres.     

人脑星形细胞肿瘤CD133和巢蛋白阳性细胞的分布特征

目的 探讨CD133 和巢蛋白阳性细胞在人脑星形细胞肿瘤中的分布特征.方法 采集不同级别星形细胞肿瘤标本31 例,免疫组织化学染色检测其胶质瘤干细胞标志物CD133 和巢蛋白表达水平,光学显微镜下观察CD133 和巢蛋白阳性细胞在不同级别星形细胞肿瘤中的分布特征.结果 不同级别肿瘤之间和同一肿瘤不同区域之间CD133 阳性细胞的分布呈现极大变异性,低级别星形细胞肿瘤完全不表达或仅单个细胞表达,高级别星形细胞肿瘤成簇表达,其特异性分布特征为CD133 阳性细胞围绕微血管分布,单个细胞贴附于血管外壁,在肿瘤周围水肿区域亦可呈散在分布而不贴附于血管外壁;簇状或巢状分布的CD133 阳性细胞均位于近血管区域,CD133 阳性细胞形态亦不尽一致,无论是新生发芽的肿瘤性血管内皮细胞还是成熟的血管内皮细胞均不表达CD133.巢蛋白阳性细胞在不同级别肿瘤之间和同一肿瘤不同区域之间具有与CD133 阳性细胞相似的分布特征,但新生发芽的肿瘤性血管内皮细胞巢蛋白表达阳性;变性肿瘤中巢蛋白阳性细胞可呈" 开屏状"分布,而CD133 阳性细胞未见类似分布.结论 CD133 和巢蛋白阳性细胞在人脑星形细胞肿瘤中呈现近微血管分布和富集于肿瘤周围水肿区域的分布特征,新生发芽的肿瘤性血管内皮细胞巢蛋白表达阳性.