Sorcin的表达影响人胶质瘤细胞对顺铂的敏感性

 目的： 研究可溶性耐药相关钙结合蛋白(sorcin)在人胶质瘤细胞对顺铂敏感性中的作用。方法：构建pSilencerTM 3.1-H1-sorcin siRNA表达质粒;质粒转染人胶质瘤U251细胞;RT-PCR和Western blotting法检测sorcin mRNA和蛋白表达的变化;MTT法检测U251细胞的生存率;Western blotting检测耐药相关蛋白的表达变化。结果：经酶切和测序鉴定,成功构建pSilencerTM3.1-H1-sorcin siRNA表达载体;将该表达载体转染U251细胞,RT-PCR和Western blotting结果显示sorcin mRNA和蛋白表达量降低（P<0.05）;MTT结果显示,抑制sorcin表达可增强U251细胞对顺铂的敏感性（P< 0.05）;同时发现抑制U251细胞的sorcin表达能降低耐药相关蛋白P-糖蛋白(P-gp)和多药耐药相关蛋白1(MRP1)的蛋白水平（P< 0.05）。结论：抑制sorcin表达增强U251细胞对顺铂的敏感性,其作用机制可能与降低耐药相关蛋白P-gp和MRP1的表达有关,提示sorcin可能与胶质瘤细胞顺铂耐药有关。     

免疫凋亡素e23sFv-Fdt-RC6对HER2阳性胶质瘤细胞U251的杀伤作用

目的:探讨靶向活性caspase-6融合蛋白对HER2阳性胶质瘤细胞系U251的促凋亡作用.方法:利用白喉毒素furin酶识别序列(Fdt)连接抗HER2单链抗体基因e23sFv和重构型caspase-6(RC6)基因,连接入pCMV载体,构建重组基因的真核表达载体pCMV-e23sFv-Fdt-RC6.脂质体转染HER2阳性胶质瘤U251细胞系,Western blot检测目的蛋白的表达.流式细胞术(FCM)检测转染后U251细胞的凋亡率,MTT法检测目的基因转染后对U251细胞增殖的影响.结果:双酶切及基因测序证实,pCMV-e23sFv-Fdt-RC6载体被成功构建.Western blot检测到转染表达载体48 h后的U251细胞中有活性caspase-6的表达.FCM检测到实验组细胞凋亡率比对照组显著增高.MTT实验观察到转染载体pCMV-e23sFv-Fdt-Rc6的U251细胞的增殖被明显抑制.结论:e23sFv-Fdt-RC6融合蛋白对HER2阳性胶质瘤细胞系U251有明显的促凋亡作用.     

DNA修复蛋白MGMT基因的克隆及转导脐血CD34+细胞后耐药特征的研究

目的 增强脐血CD34+造血细胞对化疗药物的耐药表型,探讨逆转录病毒介导的基因转移效率和耐药基因特性,以及在脐血造血干细胞保护性基因治疗中的作用和意义.方法 应用逆转录-聚合酶链反应(RT-PCR)从人肝组织中获得编码六氧甲基鸟嘌呤-DNA-甲基转移酶(O6-methylguanine-DNA-methyltransferase,MGMT)cDNA;利用基因重组技术,将其克隆于pGEM-T质粒载体并构建了逆转录病毒载体G1Na-MGMT;应用脂质体LipofectAMINE基因转移法将后者导入GP+E86和PA317病毒包装细胞,以卡氮芥1,3-Bis(2-Chloroethyl)-1-Nitrosourea(BCNU)加压筛选后的阳性克隆上清经乒乓效应后继而感染脐血CD34+细胞.应用PCR,Southern Blot,RT-PCR,Northern blot,Western Blot及MTT法检测MGMT基因在脐血CD34+细胞中的转移和表达.结果 酶切鉴定及DNA测序证实了MGMT cDNA克隆的正确性,脂质体介导方法成功将其导入病毒包装细胞,BCNU加压筛选和乒乓感染法使病毒效价达1.6×106CFU/ml,逆转录病毒载体介导的MGMT基因在脐血造血干细胞中获得了有效转移和表达.转MGMT耐药基因脐血造血干/祖细胞对BCNU的抗药性较对照组提高了4倍.结论 DNA修复蛋白MGMT基因的克隆并导入脐血造血干/祖细胞可以明显提高靶细胞的耐药性.     

芳香化酶和雌激素相关受体在人骨髓间充质干细胞中的表达

背景：骨髓间充质干细胞中可能存在芳香化酶和雌激素受体相互作用的雌激素信号途径,调控骨髓间充质干细胞生物活性。目的：观察成人骨髓间充质干细胞成骨诱导分化过程中芳香化酶和雌激素相关受体的表达。方法：将骨髓间充质干细胞分为单纯培养组和成骨诱导组,分别用低糖DMEM完全培养基和成骨诱导分化完全培养基培养,MTT检测细胞增殖能力,茜素红染色观察成骨矿化能力,qPCR和Western blot检测芳香化酶、雌激素受体α、雌激素受体β和雌激素受体相关受体α mRNA和蛋白表达,ELISA检测细胞上清液和裂解液中雌二醇水平。结果与结论：培养72 h,成骨诱导组增殖能力最强;培养21 d,成骨诱导组茜素红染色显示大量钙沉积;qPCR和Western blot结果显示成骨诱导组能促进芳香化酶和雌激素受体α mRNA和蛋白表达,抑制雌激素受体相关受体α mRNA和蛋白表达,成骨诱导组雌激素受体β mRNA和蛋白表达与单纯培养组无差异;ELISA结果显示成骨诱导组上清液中雌二醇水平高于裂解液及单纯培养组上清液中雌二醇水平。结果表明成人骨髓间充质干细胞及成骨诱导分化后能表达芳香化酶、雌激素受体α、雌激素受体β和雌激素受体相关受体α,亦能自身合成和分泌雌激素,由此产生的雌激素可能是通过相关受体对骨髓间充质干细胞成骨分化发挥作用。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

芹菜素对U251胶质瘤细胞生长及p21、p53蛋白表达的影响

目的 探讨芹菜素对U251胶质瘤细胞生长及p21、p53蛋白表达的影响.方法 常规培养人胶质瘤U251细胞,分别用0、20、40、80 μmol/L芹菜素作用于U251细胞;四甲基偶氮唑盐微量酶反应比色法(MTT法)检测U251细胞增殖抑制率;Western blot检测p53、p21蛋白表达水平.结果 40及80 μmol/L芹菜素抑制人胶质瘤细胞U251的增殖,抑制作用呈剂量和时间依赖性;随着芹菜素浓度的增加,U251中p21和p53表达呈增加趋势.结论 芹菜素可能通过上调p21和p53表达,抑制胶质瘤细胞体外生长.     

Nix和miR-520h对神经胶质瘤发生的影响及相互作用

目的　通过综合分析肿瘤标本中基因和蛋白表达,及体外生物学作用的检测,来评价胶质瘤中Nix蛋白的作用以及Nix蛋白与miR-520h的关系。 方法　选取46例胶质瘤标本,RT-PCR检测miR-520h基因的表达,Western blotting检测Nix 蛋白的表达;培养U251胶质瘤细胞,Western blotting检测Nix蛋白的表达;构建靶向Nix基因的shRNA敲除U251细胞中Nix基因（Nix-kn）, Western blotting检测Nix, IKKα, i-κBα和p-NF-κB/p65 等蛋白的表达水平; has-miR-520h抑制剂转染U251细胞,Western blotting检测Nix, IKKα, i-κBα和p-NF-κB/p65 等蛋白的表达水平。 结果　胶质瘤标本的基因和蛋白检测表明,miR-520h的高表达伴随着Nix蛋白的高表达;体外实验结果表明,与正常培养的U251相比,缺氧培养的U251细胞Nix表达较高;与Nix-kn组相比,Nix-wt组胶质瘤细胞Nix和p-NF-κB/p65表达均明显增高,而NF-κB活性抑制剂蛋白i-κBα表达降低;200 nmol/L的has-miR-520h抑制剂处理细胞后,与正常对照组比较, Nix蛋白表达显著减少,而p-NF-kappaB/p65蛋白表达显著增加。 结论　在神经胶质瘤的发生发展中miR-520h与Nix蛋白的表达相关;miR-520h可能通过促进Nix合成,从而成为一个强大的肿瘤激活剂。     

瞬时受体电位通道M8调控NCR1/NKP46通路抑制胶质瘤细胞免疫逃逸的作用研究北大核心CSCD

目的:探讨瞬时受体电位通道M8(TRPM8)对胶质瘤细胞免疫逃逸的影响,并初步探究可能的作用机制。方法:通过实时荧光定量PCR法检测人正常胶质细胞株(SVGp12)与3种胶质瘤细胞株(U251、U373、T98G)中TRPM8基因表达;将U251细胞随机分为空白对照组、TRPM8-siRNA组、NC-siRNA组、pcDNA3.1-TRPM8组和pcDNA3.1-NC组,利用脂质体分别将TRPM8-siRNA、NC-siRNA、pcDNA3.1-TRPM8和pcDNA3.1-NC转染至U251细胞中,采用实时荧光定量PCR法和Western blot检测细胞中TRPM8基因和蛋白表达,CCK-8法、集落形成实验和流式细胞术检测细胞增殖、克隆形成及凋亡情况,乳酸脱氢酶(LDH)释放法检测NK92细胞对U251细胞的杀伤作用,ELISA法检测TNF-α、IL-2、IL-6、IFN-γ水平,流式细胞术、Western blot检测细胞调节区域因子1(NCR1)与自然杀伤因子蛋白46(NKP46)的表达。结果:3种胶质瘤细胞株U251、U373、T98G中TRPM8 mRNA表达水平均显著高于正常胶质细胞株SVGp12,其中U251细胞最高;与空白对照组相比,TRPM8-siRNA组TRPM8 mRNA和蛋白的相对表达量均下调(P<0.01),细胞活性下降(P<0.05),细胞克隆形成数目减少(P<0.01),细胞凋亡率升高(P<0.01),NK92细胞对U251细胞的杀伤率提高(P<0.01),细胞上清液中TNF-α、IL-2、IL-6及IFN-γ水平均下降(P<0.01),同时,NCR1、NKP46表达也明显增加(P<0.01);与空白对照组相比,pcDNA3.1-TRPM8组TRPM8 mRNA和蛋白的相对表达量均上调(P<0.01),细胞活性升高(P<0.05),细胞克隆形成数目增加而凋亡率下降(P<0.01),NK92细胞对U251细胞的杀伤率下降(P<0.01),细胞上清液中TNF-α、IL-2、IL-6及IFN-γ水平均升高(P<0.01),NCR1、NKP46表达减少(P<0.01)。结论:抑制TRPM8表达能够调控胶质瘤微环境中免疫抑制状态,增强NK92细胞杀伤U251细胞的能力,削弱肿瘤细胞的免疫逃逸能力,这可能与调控NCR1/NKP46通路相关。     

NEDD9在神经胶质瘤中的表达及基因沉默对人胶质瘤U251细胞增殖与侵袭的影响

目的研究NEDD9在神经胶质瘤中的表达及NEDD9基因沉默对人胶质瘤U251细胞增殖与侵袭的影响。方法选取手术切除的脑胶质瘤组织36例及正常脑组织10例,采用qRT-PCR和Western blot法检测各组脑组织中NEDD9mRNA和蛋白表达情况,构建靶向NEDD9的shRNA重组慢病毒载体,将慢病毒载体转染U251细胞,应用qRT-PCR与Western blot方法检测NEDD9mRNA与蛋白表达,MTT方法检测转染后U251细胞活力的变化,Transwell实验检测细胞侵袭能力的变化。结果脑胶质瘤组织NEDD9的蛋白与mRNA表达水平显著高于正常脑组织。成功建立了稳定沉默NEDD9基因的U251细胞株,与空白对照组及阴性对照组比较,转染NEDD9-shRNA组U251细胞活力与侵袭能力显著降低(P0.01)。结论 NEDD9可能是治疗胶质瘤U251细胞的潜在靶点。     

miR-200b通过靶向CRKL抑制胶质瘤U251细胞增殖

目的研究hsa-miR-200b对人U251胶质瘤细胞增殖的影响及相关作用机制。方法将miR-200b过表达和表达沉默质粒分别稳定转染至胶质瘤U251细胞,qRT-PCR验证转染效率,CCK-8法检测U251细胞增殖水平变化,qRTPCR以及Western blot方法检测的CRKL的m RNA和蛋白表达水平。双荧光素酶报告基因实验验证miR-200b与CRKL基因3’UTR的结合位点。将CRKL过表达和表达沉默质粒载体分别稳定转染至U251胶质瘤细胞,使用CCK-8法检测U251细胞增殖水平变化。分别建立miR-200b和CRKL的稳定双转染U251细胞系,使用CCK-8法检测对各组细胞增殖水平的变化。结果与对照组相比,miR-200b过表达显著抑制胶质瘤U251细胞的增殖并且抑制CRKL在U251胶质瘤细胞的m RNA和蛋白表达;双荧光素酶报告基因实验验证了miR-200b与CRKL基因3’UTR的结合位点;CRKL过表达显著增强胶质瘤U251细胞增殖能力;CRKL过表达有效阻断了miR-200b抑制胶质瘤U251细胞增殖的作用。结论 miR-200b能够通过靶向下调CRKL抑制胶质瘤U251细胞增殖。     

miR-130b在胶质瘤对替莫唑胺耐药中的作用

目的:探讨微小RNA-130b(microRNA-130b,miR-130b)在胶质瘤细胞中的表达及其调控体外胶质瘤细胞对替莫唑胺(temozolomide,TMZ)耐药中的作用。方法:利用RT-qPCR法检测miR-130b在胶质瘤细胞株U251、SHG-44和U87中的表达水平;计算TMZ对不同胶质瘤细胞株(U251、SHG-44和U87)的半数抑制浓度(IC_(50));通过不同浓度梯度的TMZ作用于体外U251细胞,从而获得相对稳定的对TMZ耐药的U251(U251/TMZ resistance,U251/TR)细胞,计算TMZ对U251/TR细胞的IC_(50)及耐药指数(resistance factor,RF);使用miR-130b模拟物(miR-130b mimics)和miR-130b抑制物(miR-130b inhibitor)瞬时转染体外胶质瘤细胞;CCK-8法检测体外胶质瘤细胞的活力;流式细胞术检测胶质瘤细胞凋亡;通过生物信息学工具分析miR-130b可能的靶基因,萤光素酶报告基因实验测定萤光素酶活性;电泳迁移率变动分析检测核因子κB(nuclear factor-κB,NF-κB)的活性;Western blot法测定肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、Bcl-2、X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)和survivin蛋白在胶质瘤细胞中的表达水平。结果:TMZ对不同胶质瘤细胞株(U251、SHG-44和U87)的IC_(50)分别为54.8、94.8和149.6μmol/L;TMZ对U251/TR细胞的IC_(50)为(446.5±61.3)μmol/L,其RF为8.1;转染miR-130b mimics可明显增强TMZ对U251/TR细胞的生长抑制和凋亡诱导作用;转染miR-130b inhibitor可显著抑制TMZ对U251/TR细胞的生长抑制和凋亡诱导作用;萤光素酶报告基因实验验证TNF-α是miR-130b的直接作用靶点;与mimics NC组相比,转染miR-130b mimics后U251/TR细胞中的NF-κB活性及TNF-α、Bcl-2、XIAP和survivin蛋白表达均明显下调;与inhibitor NC组相比,转染miR-130b inhibitor后U251细胞中的NF-κB活性及TNF-α、Bcl-2、XIAP和survivin蛋白表达均显著上调(P0.05);NF-κB抑制剂Bay 11-7082可增强TMZ对U251/TR细胞凋亡的诱导作用。结论:miR-130b在耐药型胶质瘤细胞中表达下调,并通过靶向调控TNF-α/NF-κB通路增强TMZ对体外胶质瘤细胞的作用。     

Is Mda‐7/IL‐24 a Potential Target and Biomarker for Enhancing Drug Sensitivity in Human Glioma U87 Cell Line?

Gliomas are the most common form of primary brain tumor with the highest mortality rates. Drug resistance is a major cause of treatment failure in patients with glioma. Melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24) has been demonstrated to play an important role in drug resistance in human cancer cell lines. However, the reversing effect of mda‐7/IL‐24 on drug resistance of human glioma is not fully clear. Here, we investigated the effects of overexpression of the mda‐7/IL‐24 gene in human glioma. We established a cisplatin‐resistant U87 glioma cell line and found that mda‐7/IL‐24 was highly correlated with drug resistance. Furthermore, we investigated the apoptotic rate, intracellular accumulation of Rhodamine‐123, and expression of glutathione and P‐glycoprotein. The over‐expression of mda‐7/IL‐24 enhanced cisplatin cytotoxicity and reversal of drug resistance in glioma cells. The reversing effect of mda‐7/IL‐24 on drug resistance was induced mainly through the regulation of drug resistance‐related genes and efflux drug pumps. Thus, mda‐7/IL‐24 can be used as a promising predictive biomarker and potential therapeutic target for chemotherapy in glioma. Anat Rec, 2013. © 2013 Wiley Periodicals, Inc.     

The Fanconi anemia (FA) pathway confers glioma resistance to DNA alkylating agents

DNA alkylating agents including temozolomide (TMZ) and 1,3-bis[2-chloroethyl]-1-nitroso-urea (BCNU) are the most common form of chemotherapy in the treatment of gliomas. Despite their frequent use, the therapeutic efficacy of these agents is limited by the development of resistance. Previous studies suggest that the mechanism of this resistance is complex and involves multiple DNA repair pathways. To better define the pathways contributing to the mechanisms underlying glioma resistance, we tested the contribution of the Fanconi anemia (FA) DNA repair pathway. TMZ and BCNU treatment of FA-proficient cell lines led to a dose- and time-dependent increase in FANCD2 mono-ubiquitination and FANCD2 nuclear foci formation, both hallmarks of FA pathway activation. The FA-deficient cells were more sensitive to TMZ/BCNU relative to their corrected, isogenic counterparts. To test whether these observations were pertinent to glioma biology, we screened a panel of glioma cell lines and identified one (HT16) that was deficient in the FA repair pathway. This cell line exhibited increased sensitivity to TMZ and BCNU relative to the FA-proficient glioma cell lines. Moreover, inhibition of FA pathway activation by a small molecule inhibitor (curcumin) or by small interference RNA suppression caused increased sensitivity to TMZ/BCNU in the U87 glioma cell line. The BCNU sensitizing effect of FA inhibition appeared additive to that of methyl-guanine methyl transferase inhibition. The results presented in this paper underscore the complexity of cellular resistance to DNA alkylating agents and implicate the FA repair pathway as a determinant of this resistance.     

MiR-16 modulate temozolomide resistance by regulating BCL-2 in human glioma cells

Temozolomide (TMZ) with radiotherapy is the current standard of care for newly diagnosed glioma. However, glioma patients who are treated with the drug often develop resistance to it and some other drugs. Recently studies have shown that microRNAs (miRNAs) play an important role in drug resistance. In present study, we first examined the sensitivity to temozolomide in six glioma cell lines, and established a resistant variant, U251MG/TR cells from TMZ-sensitive glioma cell line, U251MG. We then performed a comprehensive analysis of miRNA expressions in U251MG/TR and parental cells using cancer microRNA PCR Array. Among the downregulated microRNAs was miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR in U251MG/TR and U251MG cells. The selective microRNA, miR-16 mimics or inhibitor was respectively transfected into U251MG/TR cells and AM38 cell. We found that treatment with the mimics of miR-16 greatly decreased the sensitivity of U251MG/TR cells to temozolomide, while sensitivity to these drugs was increased by treatment with the miR-16 inhibitor. In addition, the downregulation of miR-16 in temozolomide-sensitive AM38 cells was concurrent with the upregulation of Bcl-2 protein. Conversely, overexpression of miR-16 in temozolomide-resistant cells inhibited Bcl-2 expression and decreased temozolomide resistance. In conclusion, MiR-16 mediated temozolomide-resistance in glioma cells by modulation of apoptosis via targeting Bcl-2, which suggesting that miR-16 and Bcl-2 would be potential therapeutic targets for glioma therapy.     

Sensitization of malignant glioma to chemotherapy through dendritic cell vaccination

Drug resistance represents a major cause of chemotherapy failure in patients with cancer. The characterization of the molecular pathways involved in drug resistance has provided new targets to circumvent or reverse chemotherapy resistance. Many of these target proteins are often overexpressed in human glioma and have been identified as tumor antigens, which implicate the development of immunotherapy as a therapeutic strategy. Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and have been demonstrated to stimulate antibody and cell-mediated immune responses against tumor-associated antigens. Ex vivo-generated and tumor antigen-loaded DCs have been successfully introduced to clinical vaccination protocols, which have proven to be feasible and effective in some glioma patients. Most importantly, immunotherapy followed by chemotherapy could significantly increase 2-year survival in malignant glioma patients, which obviously demonstrates that DC vaccination could increase the sensitivity of tumor cells to chemotherapy. This review focuses on recent advances in the identification of tumor-associated antigen in glioma, as well as novel insights into their biological function related to drug resistance. These insights may provide the rationale for a novel strategy of a DC cancer vaccine that sensitizes tumor cells to chemotherapy. In addition, the current research status and the future direction of a DC-based vaccine to treat glioma in animal models and clinical trials will also be discussed.     

P-glycoprotein and multidrug resistance-associated protein mediate specific patterns of multidrug resistance in malignant glioma cell lines, but not in primary glioma cells

Understanding and overcoming multidrug resistance (MDR) may be a promising strategy to develop more effective pharmacotherapies for malignant gliomas. In the present study, human malignant glioma cell lines (n=12) exhibited heterogeneous mRNA and protein expression and functional activity of the mdr gene-encoded P-glycoprotein (PGP) and MDR-associated protein (MRP). Correlation between mRNA expression, protein levels and functional activity was strong. Inhibition of PGP activity by verapamil or PSC 833 enhanced the cytotoxic effects of vincristine, doxorubicin, teniposide and taxol. Inhibition of MRP activity by indomethacin or probenecid enhanced the cytotoxic effects of vincristine, doxorubicin and teniposide. The human cerebral endothelial cell line, SV-HCEC, exhibited the strongest PGP activity of all cell lines. Five primary human glioblastomas and one anaplastic astrocytoma displayed heterogenous protein levels of PGP and MRP-1 in tumor cells and of PGP in biopsy specimens in vivo, but no functional activity of these proteins upon ex vivo culturing. These data suggest that the glioma cell line-associated MDR-type drug resistance is a result of long-term culturing and that cerebral endothelial, but not glioma cells, may contribute to MDR-type drug resistance of gliomas in vivo.     

Significance of perivascular tumour cells defined by CD109 expression in progression of glioma

In the progression of glioma, tumour cells often exploit the perivascular microenvironment to promote their survival and resistance to conventional therapies. Some of these cells are considered to be brain tumour stem cells (BTSCs); however, the molecular nature of perivascular tumour cells has not been specifically clarified because of the complexity of glioma. Here, we identified CD109, a glycosylphosphatidylinositol‐anchored protein and regulator of multiple signalling pathways, as a critical regulator of the progression of lower‐grade glioma (World Health Organization grade II/III) by clinicopathological and whole‐genome sequencing analysis of tissues from human glioma. The importance of CD109‐positive perivascular tumour cells was confirmed not only in human lower‐grade glioma tissues but also in a mouse model that recapitulated human glioma. Intriguingly, BTSCs isolated from mouse glioma expressed high levels of CD109. CD109‐positive BTSCs exerted a proliferative effect on differentiated glioma cells treated with temozolomide. These data reveal the significance of tumour cells that populate perivascular regions during glioma progression, and indicate that CD109 is a potential therapeutic target for the disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Persistence of CD133+ cells in human and mouse glioma cell lines: detailed characterization of GL261 glioma cells with cancer stem cell-like properties

The concept of cancer stem cells suggests that there are malignant stem-like cells within a tumor that are responsible for tumor renewal and resistance to cytotoxic therapies. Studies have identified glioma stem-like cells that extrude Hoechst 33342 dye, representing a double-negative "side population" (SP) thought to be selectively resistant to drug therapy. A CD133+ stem cell-like subpopulation has been isolated from a human glioma that was enriched for tumor-initiating cells. It is unknown whether CD133+ cells with similar phenotype persist in established glioma cell lines, or if CD133 is a marker of glioma stem-like cells in rodents. We investigated whether CD133+ and SP cells existed in the GL261 cell line, a syngeneic mouse glioma model that is widely used for preclinical and translational research. Intracerebral injection of less than 100 CD133+ GL261 cells formed tumors, whereas it required 10,000 CD133(-) cells to initiate a tumor. CD133+ GL261 cells expressed nestin, formed tumor spheres with high frequency, and differentiated into glial and neuronal-like cells. Similar to GL261, seven human glioma cell lines analyzed also contained a rare CD133+ population. Surprisingly, we found that CD133+ GL261 cells did not reside in the SP, nor did the majority ( approximately 94%) of CD133+ human glioma cells. These results demonstrate that the expression of CD133 in murine glioma cells is associated with enhanced tumorigenicity and a stem-like phenotype. This study also reveals a previously unrecognized level of heterogeneity in glioma cell lines, exposing several populations of cells that have characteristics of cancer stem cells.     

4E-BP1在人脑胶质瘤细胞对卡氮芥获得性耐药中的作用

 目的： 探讨真核翻译起始因子4E结合蛋白1（4E-BP1）与人脑胶质瘤细胞耐药性的关系,为寻找克服胶质瘤获得性耐药的新靶点提供理论依据。方法：Western blotting 检测SWOZ2及其获得性耐药细胞株SWOZ2-BCNU中4E-BP1及其磷酸化蛋白（p-4E-BP1）的表达。小干扰RNA下调4E-BP1表达后,细胞计数试剂盒8检测SWOZ2细胞对卡氮芥（BCNU）的耐药性。免疫荧光法检测4E-BP1及p-4E-BP1(T70)蛋白在2株细胞中的表达位置。结果：SWOZ2-BCNU细胞中4E-BP1及其磷酸化蛋白表达较SWOZ2细胞明显下降。下调4E-BP1表达能明显增加敏感细胞对BCNU的耐药性。SWOZ2-BCNU细胞p-4E-BP1(T70)蛋白在获得性耐药过程中出现了核转位。结论：4E-BP1作为翻译调控关键蛋白,可能参与了人脑胶质瘤获得性耐药的发生机制。