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.     

hMena在胶质瘤中的表达及分布特征

目的 观察人脑胶质瘤中hMena蛋白的表达情况及分布特征,并分析其表达与病理分级的相关性. 方法 选择天津医科大学总医院神经外科自2008年8月至2009年12月间手术切除并经病理证实的胶质瘤标本65例(WHO Ⅰ级9例,Ⅱ级16例,Ⅲ级19例,Ⅳ级21例),其中6例胶质母细胞瘤(GBM,Ⅳ级)患者术中在神经导航系统引导下在肿瘤实体区、肿瘤与脑组织交界区和瘤周1.5 cm处取材,另取同期5例因脑出血行减压术患者的正常脑组织标本作为对照.应用免疫组化染色与Western blotting检测标本中hMena蛋白的表达情况和分布特征. 结果 免疫组化染色结果显示对照脑组织中无hMena阳性染色,胶质瘤hMena阳性染色定位于胞浆,不同级别胶质瘤中hMena的表达差异有统计学意义(x2=34.935,P=0.000),hMena的表达与胶质瘤的分级呈正相关关系(rs=0.682,P=0.000).Westemblotting检测发现不同病理分级的胶质瘤中,hMena的表达水平随病理级别升高而逐渐升高. 结论 hMena蛋白可能在胶质瘤恶性进展中发挥作用.

Abstract：

Objective To observe the expression and distribution characteristics of human orthology of mammalian enabled (hMena) in human glioma, and analyze the correlation of its expression with the pathological grade of the glioma. Methods Sixty-five specimens of glioma with different pathological grades were collected; among these samples, according to the WHO classification, grade Ⅰ was noted in 9, grade Ⅱ in 16, grade Ⅲ in 19 and grade Ⅳ in 21; in 6 of these patients with grade Ⅳ glioblastoma multiforme, tissues from the lesion, the junctional zone between the glioma and the normal brain tissue, and the surrounding area (with a diameter of 1.5 cm) were separated under the assistance of neuronavigation system. Another 5 normal brain tissues from patients with cerebral hemorrhage performed decompression were chosen as controls. Immunohistochemistry and Western blotting were used to detect the expression and distribution characteristics of hMena. Results The hMena expression was negative in control brain tissue, and its positive rates of gliomas with different grades were 11.11% (1/9), 25% (4/16), 84.21% (16/19) and 90.48% (19/21) in gliomas of grade Ⅰ, grade Ⅱ, grade Ⅲ and grade Ⅳ, respectively; and these staining was mainly located in cytoplasm; the hMena expression in gliomas with different grades was significantly different (x2=34.935, P=0.000). The expression rate of hMena was positively correlated with the increasing grade of WHO classification (rs=0.682, P=0.000).Western blotting indicated that, in gliomas with different pathological grades, hMena expression levels increased gradually with the increase of pathological grade. Conclusion The hMena protein might play a role in the malignant progression of glioma.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

人脑胶质瘤CXCL12基因的甲基化分析研究

Objective To detect the methylation status of CXCL12 gene and the mRNA expression of CXCL12, CXCR4 and DNA methyhransferases (DNMTs) in glioma, and to analyze the methylation regulation and mechanism of CXCL12/CXCR4 signaling axis in the malignant progress of glioma. Methods The mRNA expression of CXCL12, CXCR4, DNMT1, DNMT3A and DNMT3B was detected by the semi - quantitative RT - PCR and real - time PCR in 76 gliomas and 10 normal brain tissues. The methylation status of CXCL12 in glioma was also studied by the methylation specific PCR. Results ( 1 ) The mRNA expression of CXCR4 in glioma increased with WHO grades. (2) Methylation of CXCL12 was detected in 34. 2% ( 26/76 ) of gliomas, but the methylation rate decreased with WHO grades. ( 3 ) Epigenetic inactivation of CXCL12 mainly happened in low- grade gliomas, and the CXCL12 mRNA levels were closely related to its methylation status. (4) The expression levels of these three DNMT genes were significantly higher in the CXCL12 - methylated gliomas than in the CXCL12 - unmethylated ones. Conclusion The CXCR4 gene may be a marker of aggressive biological behavior of glioma. The CXCL12 promoter hypermethylation is detected mainly in low - grade gliomas. And the methylaiton of CXCL12 gene cause the down - regulation of its mRNA levels in low - grade gliomas. The high expressions of DNMT1、DNMT3A and DNMT3B may be the potential mechanism of CXCL12 methylation.     

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.     

ABCG2与VEGF、VEGFR和CD34在胶质瘤组织芯片中的共表达

目的 探讨ABCG2在胶质瘤血管形成过程中与VEGF、VEGFR和CD34的关系和共表达.方法 构建布有90例各级别人脑胶质瘤的组织芯片,用EnVision法的免疫组织化学染色,分别分析ABCG2、VEGF、VEGFR和CD34在胶质瘤中的表达率、微血管密度(MVD)及其相关性,并以免疫荧光染色和激光共聚焦显微镜检测CD34和ABCG2共表达的细胞.结果 ABCG2、VEGF、VEGFR和CD34的表达率均随胶质瘤恶性程度增加而增高;并且ABCG2表达水平与肿瘤MVD显著相关,γ=0.540,P＜0.001.ABCG2阳性表达的Ⅲ、Ⅳ级肿瘤标本MVD比Ⅰ、Ⅱ级高.VEGF、VEGFR和ABCG2均为阳性表达的肿瘤标本MVD平均值显著高于ABCG2阴性表达者,P＜0.001.在CD34显示的肿瘤血管腔壁上同时见到了ABCG2的表达.结论 胶质瘤的恶性进展除了与公认的肿瘤细胞和肿瘤血管内皮细胞共表达的VEGF和VEGFR高表达相关,还与ABCG2高表达相关.在同一根血管上同时见到了ABCG2(肿瘤干细胞标志蛋白)和CD34(内皮细胞标志蛋白)的共表达,表明肿瘤干细胞除了生成肿瘤细胞,还有可能生成肿瘤血管内皮细胞.     

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.     

脑肿瘤干细胞的增殖活性与病理级别的相关性研究

目的检测增殖细胞核标记物Ki-67与脑肿瘤干细胞（BTSCs）标记物CD133、Nestin在60例脑胶质瘤组织中的表达,探讨BTSCs的增殖活性与病理级别的相关性。方法采用免疫组织化学SP法检测CD133、Nestin和Ki-67在60例胶质瘤组织中的表达;采用免疫荧光双染法检测Ki-67、CD133和Nestin之间的共表达情况。计算两种方法中CD133＋细胞、Nestin＋细胞和Ki-67＋细胞所占的百分率,并将Ki-67＋细胞与CD133＋细胞、Nestin＋细胞和肿瘤病理分级分别进行相关性分析。结果按照WHO 2000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19例,在不同的病理级别组中,CD133＋、Nestin＋和Ki-67＋细胞的表达有明显差异,并且免疫荧光双染相比免疫组化单染更能代表BTSCs的增殖活性研究。在免疫荧光共表达中,随着病理级别的升高,CD133＋/Ki-67＋细胞（F=30.668,P=0.000）或Nestin＋/Ki-67＋细胞（F=15.316,P=0.000）的百分比差异都有显著性,并且同时均与CD133＋/Nestin＋BTSCs的表达成正相关。结论Ki-67＋指标与肿瘤级别成正相关,可以作为预后判断的指标,同时与BTSCs标记物CD133、Nestin的表达之间也有明显的相关性,并且,免疫荧光双染得出的BTSCs的增殖活性与病理级别的相关性研究更有统计学意义。     

内皮抑素和血管生成抑素融合基因修饰的单纯疱疹病毒对胶质瘤干细胞的体外实验研究

目的 探讨内皮抑素和血管生成抑素( Endo - Angio)融合基因修饰的单纯疱疹病毒(VAE)对胶质瘤干细胞(GSCs)的体外溶瘤作用。方法 新鲜高级别（WHOⅢ级、Ⅵ级）人脑胶质瘤标本经原代培养获得GSCs,采用流式细胞术检测其中CD133阳性细胞数,单克隆形成实验检测GSCs的自我更新能力,免疫荧光技术检测未分化GSCs的Nestin及分化后GFAP、NF和MAG的表达;MTS法检测VAE对GSCs增殖活性的影响,RT - PCR和Western blot检测Endo - Angio融合蛋白的表达并检测其对人脑微血管内皮细胞(HBMEC)增殖的影响;最后观察病毒处理后仍存活细胞的自我更新和诱导贴壁情况。结果 (1)从20例高级别胶质瘤标本中培养出4例GSCs,能够表达CD133及Nestin,具有自我更新能力和多向分化能力。(2)VAE能够感染GSCs,4例GSCs增殖活性明显下降(P＜0.05)。(3)VAE感染GSCs 48 h后,基因水平及蛋白水平都有Endo- Angio融合蛋白表达,该融合蛋白使HBMEC增殖活性明显下降(P＜0.05)。(4)VAE感染后仍存活的细胞不再具有形成GSCs球的能力,加入血清诱导后也不再贴壁生长。结论 在体外,VAE能够显著抑制GSCs活性,能够表达具有生物活性的Endo-Angio融合蛋白,为脑胶质瘤溶瘤病毒治疗开辟了新的途径。     

人脑胶质瘤中CD133、SSEA-1、Nestin的表达和意义

目的：研究胶质瘤干细胞标志物CD133、SSEA-1和Nestin在胶质瘤组织中表达及其与病理分级的相关性;探讨三者在人脑胶质瘤的诊断及恶性程度判断中的临床意义。方法应用免疫组织化学方法检测54例脑胶质瘤组织和6例正常脑组织标本中CD133、SSEA-1及Nestin的表达。结果 CD133、SSEA-1和Nestin在胶质瘤组织中的阳性细胞平均表达率分别为25.38%、26.62%和22.39%,而在正常脑组织中均无表达。CD133、SSEA-1和Nestin阳性细胞率在胶质瘤各病理级别间比较,差异均有统计学意义,且三者的表达与胶质瘤病理级别呈正相关（P＜0.05）。SSEA-1与CD133、CD133与Nestin及SSEA-1与Nestin阳性细胞表达均呈正相关（P＜0.05）。结论检测CD133、SSEA-1、Nestin表达,有利于胶质瘤的诊断及恶性程度判断,并在胶质瘤的个性化综合治疗和预后评估发挥作用。     

胶质瘤组织中Nanog基因的表达及其意义

目的探讨Nanog基因在不同病理级别胶质瘤组织中的表达及其意义。方法采用免疫组织化学染色法和逆转录酶-聚合酶链反应（RT-PCR）方法从蛋白和mRNA水平检测50例不同病理级别胶质瘤组织和7例正常脑组织标本中Nanog的表达。结果 Nanog蛋白在胶质瘤组织WHOⅡ级、Ⅲ级、Ⅳ级的阳性细胞率分别为18.3%±9.6%、37.9%±19.0%、53.4%±19.8%,差异有统计学意义（F=14.918,P=0.000）;NanogmRNA的相对含量在胶质瘤组织WHOⅡ级、Ⅲ级、Ⅳ级中分别为0.1824±0.0310、0.3730±0.0961、0.4594±0.0453,差异有统计学意义（F=65.901,P=0.000）,Nanog蛋白和mRNA表达强度随着病理级别增高而升高,而在正常脑组织中均未见表达。结论 Nanog在胶质瘤组织中高表达,其表达强度与病理级别密切相关,为进一步研究其与肿瘤干细胞关系及其在胶质瘤的生物学行为中所发挥的作用奠定基础。     

人脑胶质瘤EPO与CD105表达及意义

目的 探讨人脑胶质瘤组织促红细胞生成素（EPO）和CD105的表达变化及其意义。方法 收集2002~2008年人脑胶质瘤标本152例,其中WHO分级Ⅰ级4例,Ⅱ级32例,Ⅲ级68 例,Ⅳ级48例;取同期颅脑损伤内减压术切除正常脑组织20例为对照,采用免疫组化染色分析EPO及CD105表达。收集2005~2008年人脑胶质瘤标本胶质瘤17例（WHO Ⅱ级6例,Ⅲ~Ⅳ级11例）,正常对照5例,采用实时荧光定量PCR检测EPO mRNA表达变化。术后随访截止2010年4月23日,应用Kaplan-Meier生存曲线分析高级别（Ⅲ~Ⅳ级）胶质瘤生存曲线。结果 胶质瘤EPO表达阳性率（60.5%,92/152）明显高于正常脑组织（10%,2/20;P<0.001）,胶质瘤EPO表达强度与病理分级呈正相关（rs=0.368,P<0.001）。EPO表达阳性组CD105阳性率明显高于阴性组（P<0.05）,EPO高表达组明显高于低表达组（P<0.05）。Ⅱ级胶质瘤组EPO mRNA表达水平明显高于正常组与Ⅲ~Ⅳ级胶质瘤组（P<0.05）。对于高级别（WHO Ⅲ~Ⅳ级）胶质瘤,EPO低表达组中位生存时间为12个月,高表达组为36个月;EPO低表达组累积生存率明显低于高表达组（P<0.05）。结论 人脑胶质瘤EPO蛋白的表达与病理级别及新生血管正相关;WHO Ⅱ级胶质瘤EPO mRNA在转录水平已上调;WHO Ⅲ~Ⅳ级组胶质瘤EPO表达高者生存期长。     

Stathmin／Oncoprotein18（Op18）在人脑胶质瘤中的表达研究

目的探讨Stathmin／oneoprotein18（Op18）基因在人脑胶质瘤中的表达规律及意义。方法分别采用免疫组化法和Westernblot法检测10例正常脑组织和56例脑胶质瘤中Stathmin蛋白的表达。结果免疫组化法检测Stathmin蛋白在正常脑组织、低级别胶质瘤（Ⅰ～Ⅱ级）及高级别胶质瘤（Ⅲ～Ⅳ级）中阳性表达率分别为20％、65％、100％。正常脑组织分别与Ⅰ～Ⅱ级组、Ⅲ～Ⅳ级组比较，均有显著性差异（P〈0．05）；Ⅰ～Ⅱ级组与Ⅲ～Ⅳ级组比较，有显著性差异（P〈0．05）。Westernblot法检测显示，Stathmin蛋白在胶质瘤中的表达明显增高，正常脑组织分别与Ⅰ～Ⅱ级组、Ⅲ～Ⅳ级组比较，差异均有显著性（P〈0．01），Ⅰ～Ⅱ级组与Ⅲ～Ⅳ级组比较，差异有显著性（P〈0．01）。结论Stathmin在脑胶质瘤中过表达，Stathmin可能为脑胶质瘤的生物治疗提供一个新靶点。