脑胶质瘤中hMLH1基因表达及启动子区的甲基化状态研究

目的检测脑胶质瘤中hMLH1基因表达及其启动子区的甲基化状态,探讨其在肿瘤发生中的作用。方法采用免疫组化及甲基化特异性聚合酶链反应方法,检测51例脑胶质瘤hMLH1基因表达和启动子区甲基化状态。结果 hMLH1阳性表达率为78.4%(40/51),甲基化率为13.7%(7/51),均与性别、年龄、病理类型无相关性。hMLH1在低级别(Ⅰ~Ⅱ级)胶质瘤和高级别(Ⅲ~Ⅳ级)胶质瘤中的阳性表达率分别为92.0%(23/25)和65.4%(17/26),两者差异显著(P0.05)。hMLH1在低级别(Ⅰ~Ⅱ级)和高级别胶质瘤中hMLH1启动子区甲基化率分别为0.0%(0/25)和26.9%(7/26),两者差异显著(P0.05)。hMLH1表达阳性的胶质瘤和表达阴性的胶质瘤中启动子区甲基化率分别为7.5%(3/40)和36.4%(4/11),两者差异显著(P0.05)。结论 hMLH1基因在胶质瘤中表达及启动子区甲基化状态与胶质瘤病理分级有关,hMLH1可能在胶质瘤的发展中起作用,其启动子区甲基化可能负调控hMLH1基因表达。     

人脑胶质瘤CXCL12基因的甲基化分析研究

目的 检测胶质瘤CXCL12基因启动子区的甲基化状态及其mRNA表达水平,以及受体CXCR4、DNA甲基转移酶等基因的mRNA表达情况,分析甲基化在CXCL12/CXCR4生物轴参与胶质瘤恶性进展中的调控机制.方法 半定量RT-PCR和实时定量PCR检测CXCL12、CXCR4、DNMT1、DNMT3A和DNMT3B基闪在76例胶质瘤及10例正常脑组织中的表达情况;甲基化PCR检测CXCL12基因启动子区的甲基化状态.结果 (1)CXCR4 mRNA随胶质瘤恶性程度的增高而表达增加;(2)CXCL12基因在胶质瘤中的甲基化率为34.2%,甲基化率随胶质瘤恶性程度的增高而降低;(3)CXCL12基因的甲基化主要发生在低度恶性胶质瘤中,其甲基化状态与mRNA表达密切相关;(4)DNMT1、DNMT3A和DNMT3B在CXCL12基因甲基化胶质瘤中的表达明显高于未发生甲基化的胶质瘤.结论 CXCR4基因有望成为胶质瘤恶性程度的生物学标志;CXCL12基因启动子区的甲基化主要发生在低度恶性胶质瘤中,其CXCL12基因甲基化下调mRNA的表达;DNMT1、DNMT3A和DNMT3B的过表达可能参与CXCL12基因甲基化的调控.     

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.     

人脑胶质瘤中CCND1基因的转录与表达

目的检测和分析人脑胶质瘤中CCND1基因的转录与表达,及其对肿瘤发生、发展的生物学意义.方法采用免疫组化、原位杂交法检测52例人脑胶质瘤及8例正常人脑组织中Cyclin D1 mRNA蛋白以及增殖细胞核抗原(PCNA)的表达水平.结果脑胶质瘤中CD1蛋白及mRNA呈过度表达,其标记指数和阳性率随肿瘤的恶性程度增高而增加.两者的标记指数之间、以及与PCNA标记指数之间均为显著正性相关.结论人脑胶质瘤中CCND1过度转录和表达,随胶质瘤临床病理分级增加而增高,同时与肿瘤细胞增殖状态密切相关,提示CCND1基因的异常转录和表达在胶质瘤的发生与发展中起着重要的促进作用.     

特异性shRNA干扰人慢病毒载体抑制U251细胞株Chk1、Chk2基因的表达

目的构建针对细胞周期检测点激酶1和2(Chk1和Chk2)基因的短发夹RNA(shRNA)表达载体,包装成慢病毒,建立稳定转染的细胞株,为探讨抑制Chk1和Chk2基因表达对脑胶质瘤细胞生物学行为调控的研究奠定基础。方法根据GenBank数据库提供的Chk1和Chk2基因核苷酸序列,选择设计2条能转录短发夹RNA(shRNA)的DNA序列,命名为Chk1-shRNA和Chk2-shRNA,同时设计1条非特异性序列作为阴性对照,命名为blank-shRNA。并与pLKO.1-TRC质粒载体连接,转化感受态大肠杆菌,挑取阳性克隆,抽取重组质粒,使用限制性内切酶EcoRⅠ、NcoⅠ酶切电泳,DNA测序鉴定,包装慢病毒。3组重组表达慢病毒载体转染胶质瘤细胞系U251,用嘌呤霉素筛选后挑选单克隆并扩增获得稳定株。逆转录酶-聚合酶连反应(RT-PCR)和Western blot分别在mRNA和蛋白水平上检测Chk1和Chk2的表达。结果重组质粒成功转化感受态大肠杆菌,经酶切琼脂糖凝胶电泳分析,结果表明寡核苷酸成功插入到预计位点,经测序鉴定,序列完全正确。嘌呤霉素对U251细胞的筛选浓度为4ug/ml,筛选出稳定转染三种质粒的U251细胞,Chk1-shRNA和Chk2-shRNA组细胞各自Chk1和Chk2的mRNA和蛋白表达水平明显低于blank-shRNA组。结论成功构建了针对Chk1和Chk2基因的shRNA慢病毒表达载体,转染后可抑制ChK1和Chk2基因的表达,为进一步研究Chk1和Chk2基因在脑胶质瘤细胞中的作用奠定了基础。     

慢病毒干扰Chk1、Chk2表达对脑胶质瘤系U87细胞的放射治疗敏感性的影响

目的 探讨慢病毒干扰细胞周期检测点激酶1、2(Chk1、Chk2)基因表达对脑胶质瘤细胞放射治疗敏感性的影响.方法 根据基因库数据提供的Chk1及Chk2基因核苷酸序列,各选择设计一条适合转录短发夹RNA(shRNA)的DNA序列,构建慢病毒并转染胶质瘤细胞系U87细胞进行传代扩增.用实时PCR和免疫印迹法分别在mRNA和蛋白水平上测定Chk1及Chk2的表达.转染后U87胶质瘤细胞经X线照射48 h后,进行细胞周期和凋亡的检测.结果 成功包装慢病毒后转染U87细胞,实时PCR检测Chk1和Chk2的干扰效应,其抑制率分别为73.74%和85.12%.细胞周期检测显示照射组和照射干扰Chk2组的细胞大部分阻滞在G2/M期;照射干扰Chk1组细胞放射治疗后的凋亡率较对照组和干扰Chk2组细胞明显增高(P<0.05).结论 慢病毒转染细胞可以有效的将干扰Chk1和干扰Chk2基因带到U87细胞并发挥作用,干扰Chk1和Chk2可以增加U87细胞对放射治疗的敏感性,为胶质瘤的治疗提供了新的治疗途径.     

靶向沉默Wip1基因表达对胶质瘤细胞增殖及放疗敏感性的作用

目的 探讨靶向沉默Wip1基因表达对脑胶质瘤细胞增殖及放疗敏感性的影响。方法 体外培养人胶质瘤细胞株U251,用携带Wip1基因RNA干扰载体的慢病毒感染U251细胞沉默Wip1基因表达,然后对U251胶质瘤细胞进行放疗干预。根据对U251细胞处理方法分为4组:Wip1基因沉默后放疗组（IR+Wip1组）、单纯Wip1基因沉默组（Wip1组）、单纯放疗组（IR组）和NC组（空载体病毒感染U251细胞）。CCK-8法检测细胞的增殖,实时定量PCR检测细胞Chk1、Chk2 mRNA表达,免疫印迹检测细胞Chk1、Chk2蛋白表达。结果 U251细胞慢病毒感染后3~4 d,采用荧光显微镜观察,根据绿色荧光蛋白阳性表达判定感染效率,结果显示感染效率均90％以上。放疗后24、48、72、96、120 h,IR组和Wip1组增殖率均显著低于NC组（P<0.05）,而IR+Wip1组细胞增殖率明显低于IR组和Wip1组（P<0.05）。放疗后24 h,IR+Wip1组Chk1 mRNA表达明显低于Wip1组（P<0.05）,明显高于IR组和NC组（P<0.05）;IR+Wip1组Chk2 mRNA表达明显低于其他3组（P<0.05）。IR+Wip1组Chk1蛋白表达明显低于IR组和NC组（P<0.05）,Chk2蛋白表达低于其他3组（P<0.05）。结论 靶向沉默 Wip1 基因表达可抑制胶质瘤细胞的增殖,并可能通过抑制 Chk1 、 Chk2 的蛋白表达增加胶质瘤细胞对放疗的敏感性。     

Chk1 Inhibition Ameliorates Alzheimer’s Disease Pathogenesis and Cognitive Dysfunction Through CIP2A/PP2A Signaling

Alzheimer’s disease (AD) is the most common neurodegenerative disease with limited therapeutic strategies. Cell cycle checkpoint protein kinase 1 (Chk1) is a Ser/Thr protein kinase which is activated in response to DNA damage, the latter which is an early event in AD. However, whether DNA damage-induced Chk1 activation participates in the development of AD and Chk1 inhibition ameliorates AD-like pathogenesis remain unclarified. Here, we demonstrate that Chk1 activity and the levels of protein phosphatase 2A (PP2A) inhibitory protein CIP2A are elevated in AD human brains, APP/PS1 transgenic mice, and primary neurons with Aβ treatment. Chk1 overexpression induces CIP2A upregulation, PP2A inhibition, tau and APP hyperphosphorylation, synaptic impairments, and cognitive memory deficit in mice. Moreover, Chk1 inhibitor (GDC0575) effectively increases PP2A activity, decreases tau phosphorylation, and inhibits Aβ overproduction in AD cell models. GDC0575 also reverses AD-like cognitive deficits and prevents neuron loss and synaptic impairments in APP/PS1 mice. In conclusion, our study uncovers a mechanism by which DNA damage-induced Chk1 activation promotes CIP2A-mediated tau and APP hyperphosphorylation and cognitive dysfunction in Alzheimer’s disease and highlights the therapeutic potential of Chk1 inhibitors in AD.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-022-01204-z.     

E2F1蛋白在胶质瘤中的表达及其对胶质瘤细胞生长的影响

目的探讨E2F1蛋白在胶质瘤中的表达及其对胶质瘤细胞生长的影响。方法用实时荧光定量PCR法检测E2F1mRNA在各级别胶质瘤组织中的表达水平。应用RNA干扰技术降低E2F1表达后,采用Western印迹法验证E2F1蛋白的变化。采用四甲基偶氮唑盐(MTT)微量酶反应比色法和糖检测分析试剂盒检测干扰E2F1表达后胶质瘤细胞的增殖和糖代谢的变化。结果胶质瘤组织中E2F1的表达高于正常脑组织,其表达随着级别的增高而升高;抑制E2F1的表达后,U87细胞生长受抑制,细胞的葡萄糖消耗量明显降低。结论 E2F1在胶质瘤中的表达随着肿瘤恶性程度的增高而升高;抑制E2F1表达可以明显抑制胶质瘤细胞增殖及代谢能力。     

Skp2、P27kip1和PTEN在人脑胶质瘤中的表达及其相互关系的研究

目的探讨S期激酶相关蛋白2（skp2）、细胞周期素依赖性激酶抑制蛋白P27^kip1和抑癌基因蛋白PTEN表达与人脑胶质瘤发生、发展的关系。方法用免疫组化SP法检测52例人脑胶质瘤和8例正常脑组织标本中Skp2、P27^kip1和PTEN蛋白的表达。结果Skp2在8例正常脑组织中全部表达阴性。在52例胶质瘤标本中随着肿瘤恶性程度增高而表达增强。在低、高级别胶质瘤中的阳性率分别56％和81．5％，三者之间的表达水平差异有显著性意义（P〈0．001）。P27^kip1和PTEN在正常脑组织中表达均为强阳性，随着肿瘤恶性程度增高而表达减弱，P27^kip1和PTEN各自在低、高级别人脑胶质瘤中阳性率分别96％、77．8％和80％、55．6％。三者之间的表达水平差异有显著性意义（P〈0．01）。在52例胶质瘤标本中，Skp2表达与P27^kip1（r=-0．490，P〈0．01）和肿瘤抑制蛋白PTEN（r=-0．489，P〈0．01）表达呈负相关，PTEN表达与P27^kip1表达呈正相关（r=-0．802，P〈0．01）。结论人脑胶质瘤中Skp2蛋白过表达与P27^kip1降解及PTEN蛋白低表达有关，提示Skp2蛋白过表达可能是人脑胶质瘤发生和发展的一个重要原因。     

ICAM-1、MMP-2在人脑胶质瘤的表达及相关性研究

目的 探讨细胞间黏附分子-1 (ICAM-1)和基质金属蛋白酶2(MMP-2)在人脑胶质瘤中的表达及相关性. 方法 选择郑州大学第五附属医院神经外科自2010年3月20日至2014年5月10日手术切除的胶质瘤组织标本60例,按WHO中枢神经系统肿瘤组织学分级分为Ⅰ～Ⅱ级26例(低度恶性胶质瘤组)、Ⅲ～Ⅳ级34例(高度恶性胶质瘤组),另取同期因脑外伤行内减压术患者的正常脑组织10例作为对照组,应用反转录-聚合酶链反应(RT-PCR)和免疫组化染色分别检测胶质瘤正常脑组织标本ICAM-1、MMP-2 mRNA及蛋白的表达. 结果 对照组、低度恶性胶质瘤组、高度恶性胶质瘤组标本中ICAM-1、MMP-2 mRNA的表达均依次增加,差异有统计学意义(P＜0.05);对照组、低度恶性胶质瘤组、高度恶性胶质瘤组标本中ICAM-1蛋白表达阳性率依次增高,分别为20％、46.1％、88.2％;MMP-2蛋白表达阳性率依次增高,分别为10％、50％、91.1％,比较差异有统计学意义(P＜0.05);人脑胶质瘤组织ICAM-1 mRNA和MMP-2 mRNA的表达存在正相关关系(r=0.702,P=0.001). 结论 ICAM-1、MMP-2的表达与胶质瘤恶性程度关系密切,随病理级别的增高表达水平增高;二者可能协同参与了胶质瘤中的发生及演变.     

脑胶质瘤组织中BMP-7与YY1 mRNA表达的相关性研究

目的探讨骨形态形成蛋白-7（BMP-7）和Yin Yang-1（YY1）mRNA在不同级别脑胶质瘤组织中的表达及其生物学意义。方法采用定时荧光定量PCR技术检测56例胶质瘤组织及10例正常脑组织中BMP-7和YY1mRNA的表达水平,并分析其与胶质瘤病理学分级的关系。结果①BMP-7mRNA在胶质瘤标本中的表达量明显高于正常脑组织（P〈0.01）,并且其表达量随胶质瘤恶性程度的升高而增加（P〈0.01）;②YY1 mRNA在胶质瘤标本中的表达量明显高于正常脑组织（P〈0.01）,并且其表达量随胶质瘤恶性程度的升高而增加（P〈0.01）;③BMP-7和YY1mRNA表达量在胶质瘤中的表达呈正相关（r=15.831,P〈0.01）。结论BMP-7和YY1 mRNA在胶质瘤组织中表达明显升高且两者表达均与胶质瘤的病理分级呈正相关。这提示BMP-7和YY1 mRNA的表达上调可能参与了胶质瘤的发生和发展,BMP-7和YY1可能能作为胶质瘤治疗的新靶点。     

NDRG1基因在胶质瘤中的表达及意义

目的 分析NDRG1基因在人脑胶质瘤组织中的表达情况. 方法 收集北华大学附属医院和第四军医大学西京医院神经外科自2006年2月至2007年6月手术治疗的83例胶质瘤患者的瘤组织(Ⅰ级19例、Ⅱ级22例、Ⅲ级25例、Ⅳ级17例)及12例正常脑组织,应用实时荧光定量PCR和蛋白印迹(Western blot)检测脑组织中NDRG1 mRNA和NDRG1蛋白的表达水平.结果胶质瘤组织中NDRG1 mRNA表达量和NDRG1蛋白表达量均明显低于正常脑组织,且PCR结果显示除Ⅱ、Ⅲ级比较无明显变化外,分级从Ⅰ级到Ⅳ级逐渐上升的过程中,NDRG1的表达逐渐下降,差异有统计学意义(P＜0.05). 结论 NDRG1在胶质瘤组织中呈低表达,而且其表达高低与胶质瘤的分级有关,提示其对胶质瘤的发生或发展有重要作用.     

EphA2、EphrinA1表达与脑胶质瘤恶性表型的关系

目的 探讨酪氨酸激酶受体EphA2和其配体EphrinA1表达与脑胶质瘤恶性表型的关系,为发现恶性脑胶质瘤治疗新靶点提供理论依据.方法 构建针对EphA2的RNAi载体pSilencer-EphA2-SR质粒并转染U251、U87和SHG44细胞,应用Western blotting检测转染后细胞EphA2、EphrinA1的表达,MTT法和软琼脂克隆形成实验检测细胞增殖能力的变化,体外侵袭实验和流式细胞仪分别检测细胞侵袭和凋亡的变化;建立U251、U87和SHG44细胞的裸鼠胶质瘤移植模型,分别于瘤内注射脂质体+pSilencer空载体或脂质体+pSilencer-EphA2-SR质粒,30d后比较移植瘤的平均体积和质量,免疫组化染色检测移植瘤组织EphA2和EphrinA1的表达.结果 成功构建EphA2 RNAi载体pSilencer-EphA2-SR质粒.转染pSilencer-EphA2-SR后48 h Western blotting结果显示3种细胞EphA2表达水平均降低,而EphrinA1表达水平升高;MTT、软琼脂克隆形成实验、体外侵袭实验结果显示3种细胞的A值、克隆形成率、侵袭率均低于转染pSilencer空载体者,差异有统计学意义(P＜0.05);而流式细胞仪检测显示3种细胞的凋亡率均高于转染pSilencer空载体者,差异有统计学意义(P＜0.05).瘤内注射脂质体+pSilencer-EphA2-SR质粒后移植瘤的体积和重量均低于注射脂质体+pSilencer空载体者,差异有统计学意义(P＜0.05).免疫组化染色显示瘤内注射脂质体+pSilencer-EphA2-SR质粒后EphA2表达降低,EphrinA1表达水平升高,瘤内注射脂质体+pSilencer空载体后二者的表达无明显变化.结论 EphA2、EphrinA1是脑胶质瘤恶性表型的重要调控分子,干扰EphA2分子可显著上调EphrinA1表达、部分逆转胶质瘤细胞恶性表型,提示EphA2、EphrinA1有望成为脑胶质瘤治疗新靶点.

Abstract：

Objective To investigate the relation between altered expressions of EphA2 receptor tyrosine kinase and its ligand EphrinA1 and malignant phenotype of glioma cells, and explore the new targeting molecule for treating malignant gliomas. Methods The RNAi vector (plasmid pSilencer-EphA2-SR) against EphA2 was constructed and transfected into the malignant glioma cells (U251, U87 and SHG44). Forty-eight h after the transfection, the expressions of EphA2 and EphrinA1 were detected by Western blotting; the viability and anchorage independence of the glioma cells were observed by MTT assay and colony formation assay; the invasion viability and apoptosis of glioma cells were observed by modified Boyden chamber assay and flow cytometry. Glioma xenograft models were established in nude mice; plasmid pSilencer-EphA2-SR or pSilencer-null mixed with lipofectamine was intratumorally injected. The average tumor volume and weight of the transplanted gliomas were measured and compared; the expressions of EphA2 and EphrinA1 were detected by immunohistochemical staining.Results The RNAi plasmid against EphA2 was successfully constructed. After the transfection of plasmid pSilencer-EphA2-SR, the expression of endogenous EphA2 was suppressed, which consequently resulted in increased EphrinA1 level. As compared with cells of the plasmid pSilencer-null group, cells of the plasmid pSilencer-EphA2-SR group showed obviously decreased cell viability, anchorage independence rate and in vitro invasion ability, but significantly increased cell apoptosis rate (P＜0.05).Furthermore, suppression of EphA2 resulted in delayed tumor growth and increased EphrinA 1 expression in mice xenografts. Conclusion EphA2 and EphrinA1 may be key coupled-regulators for malignant phenotype of glioma cells. EphA2 suppression partially reverses the malignant phenotype of aggressive gliomas, possibly through up-regulating the EphrinA1 expression, which indicates that EphA2 and EphrinA1 might become the new targeting molecule for treatment of gliomas.