【作者】 肖宏；

【导师】 黄强；

【作者基本信息】 苏州大学 ， 神经外科学， 2008， 博士

【摘要】 第一部分干细胞标志蛋白ABCG2与血管形成相关蛋白在组织芯片中表达关系分析目的:ABCG2已作为肿瘤干细胞标志物在研究,已知与肿瘤耐药相关,尚未知是否与肿瘤血管形成相关。本文旨在探讨其在胶质瘤血管形成过程中与VEGF、VEGFR和CD34的关系,证明其在血管形成中起主要作用。方法:构建布有90例各级别人脑胶质瘤临床标本的组织芯片,用EnVision法的免疫组化染色检测和分析ABCG2、VEGF和VEGFR(flt-1)在胶质瘤中的表达率,并以CD34单克隆抗体显示血管内皮细胞,根据CD34阳性的血管计数判定微血管密度(MVD),另用6例正常脑组织标本作为对照。结果:随着胶质瘤恶性程度的增加,ABCG2、VEGF、VEGFR(flt-1)和CD34阳性表达率均增高。ABCG2除Ⅰ、Ⅱ级之间无统计学差异外,其余各级别之间存在统计学差异(P＜0.05);Spearman等级相关分析显示ABCG2与病理级别呈正相关;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(flt-1)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。结论:在布有不同恶性程度胶质瘤标本的同一张组织芯片上证明了目前公认的肿瘤细胞诱导肿瘤血管生成的代表性基因VEGF、VEGFR和CD34与新发现的只有干细胞才表达的基因ABCG2具有统计学上的相关性,为ABCG2阳性表达细胞具有分化成肿瘤细胞和肿瘤血管细胞的多潜能研究找到了分子依据。第二部分干细胞标志蛋白ABCG2与血管形成相关蛋白在组织芯片中共表达分析目的:在明确了干细胞标志蛋白ABCG2与血管形成相关蛋白呈正相关的基础上,进一步明确是否有共表达的细胞,为进一步研究胶质瘤干细胞为胶质瘤血管起源细胞提供图像依据。方法:采用免疫荧光激光扫描共聚焦技术,观察胶质瘤组织芯片中存在的ABCG2、VEGF、CD34、vWF、GFAP共表达的细胞。结果:胶质瘤血管中存在ABCG2和CD34、vWF共表达细胞与VEGF和CD34共表达的细胞;虽然正常脑组织中也存在ABCG2与CD34、vWF,或VEGF与CD34共表达细胞,但共表达血管数量不多;在肿瘤组织和正常脑组织中均未见ABCG2和GFAP共表达细胞。结论:干细胞标志蛋白ABCG2与血管内皮细胞标志蛋白CD34和vWF共表达,不与GFAP共表达。而CD34又与VEGF共表达,说明胶质瘤干细胞还具有向胶质瘤血管内皮细胞分化的能力。这种分化还与细胞分泌VEGF因子相关,但与GFAP表达无关。第三部分ABCG2与nestin共表达细胞参与胶质瘤血管形成目的:在胶质瘤干细胞研究中已经见到nestin与干细胞标志基因CD133的关系密切,本文探索它与另一个干细胞标志基因ABCG2的关系,旨在寻找两者共表达细胞在胶质瘤血管生成中的证据。方法:从90例常规石蜡包埋、H-E染色的胶质瘤切片标本中,挑选血管最丰富,恶性程度最高的标本,进行nestin和ABCG2免疫荧光双重染色,在激光扫描共聚焦显微镜下扫描显像,成像后对两者共表达细胞与血管的关系进行分析。结果:见到了一部分组成胶质瘤血管的细胞共表达nestin和ABCG2,并有明显的血管之间的异质性。其中nestin不仅在胶质瘤血管内皮细胞有表达,还在远离血管的肿瘤细胞中也见到表达。但未见nestin和GFAP在胶质瘤血管细胞中的共表达。结论:胶质瘤干/祖细胞不但具有向亲本肿瘤细胞分化潜能和表型,还具备向肿瘤血管细胞分化的潜能和表型,为肿瘤血管形成新理论:拟态血管和马赛克血管找到了依据。第四部分ABCG2可作为高风险因子影响胶质瘤患者生存期目的:既然第一至第三部分论证了ABCG2是胶质瘤血管生成的关键基因,那么是否与患者生存预后相关就值得关注。本部分就此进行相关分析研究。方法:用第一,第二部分相同的胶质瘤组织芯片进行EnVision免疫组化染色,检测和分析ABCG2与PCNA、P53、VEGF、IGFBP-2、C-MYC在胶质瘤组织中的表达,並采用COX比例风险模型逐步回归分析上述多基因与患者生存期的关系。结果:单因素分析显示,病理分级、ABCG2、P53和VEGF等因素与患者生存期有关(P＜0.05)。而多因素分析显示切除范围、病理分级、ABCG2、P53和VEGF高表达是影响患者生存期的高风险因子。结论:ABCG2以高危因子的身份影响患者生存,可为把它作为靶分子治疗对象的进一步研究提供依据。更多还原

【Abstract】 PartⅠThe correlation of expressions of stem cell marker ABCG2 with angiopoiesis associated proteins in glioma tissues analyzed by tissue-microarray.Object:The ABCG2 is regarded as the marker of tumor stem cells,and reported to be associated with tumors’ multidrug-resistance.However,it is still unknown whether it is related to the neovascularization.In this study,we aim to explore the correlations of ABCG2 and VEGF,VEGFR and CD34 during the process of angiopoiesis in glioma,and to prove the importance of ABCG2 to angiopoiesis.Methods:A tissue microarray containing 90 specimens that include all pathological grades of human brain glioma tissues was constructed.The expressing of ABCG2、VEGF and VEGFR(flt-1) in brain tumor was detected by EnVision immunohistochemical staining.CD34 monoclonal antibody was used to label vascular endothelial cells and tumor microvasculars density(MVD) was evaluated according to the numbers of CD34 positive labeled microvasculars.Six normal brain tissues were employed as control.Results:The positive expression rates of ABCG2,VEGF,VEGFR and CD34 increased with the pathological grades.The expression of ABCG2 were statistically different(P＜0.05) among each pathological grades of gliomas except the gradeⅠ—Ⅱ.Spearman rank-correlation analysis showed the positive correlation between the expression of ABCG2 and pathological grades;moreover,the expression level of ABCG2 was notably related with MVD in tumors,γ=0.540,P＜0.001.The MVD in gliomas of gradeⅢ—Ⅳwere higher than that in gradeⅠ—Ⅱ.The genes of VEGF, VEGFR(flt-1) and CD34 were also analysized,which showed that the average value of MVD in tumors expressing VEGF~+,VEGFR~+(flt-1) and ABCG2~+ was significantly higher than that in the ABCG2~- tumors,P＜0.001.Conclusions:VEGF,VEGFR and CD34 have been generally recognized as the representative genes inducing the angiogenesis in tumors,while ABCG2 is a novel gene which only expresses in stem cells.The results of tissue-array containing glioma specimens of different pathological grades indicated their intimate the correlations.The results also suggested that the ABCG2~+ cells had the multipotential to differentiate into tumor cells and tumor vessel cells,which explored a new direction for the research on vasculogenic mimicry in tumor.PartⅡThe co-expressions of stem cell marker ABCG2 with angiopoiesis associated proteins in glioma tissue microarray.Object:The study aimed to detecte the co-expression of ABCG2 and angiopoiesis associated proteins on base of direct correlation between them,and further to supply the research of glioma stem cells’ transformation into angiocellulars in gliomas with morphological evidence.Methods:The co-expression of ABCG2、VEGF、CD34、vWF and GFAP in human glioma tissues by tissue-microarray was detected by laser scanning confocal microscope(LSCM).Results:There are co-expression cells of CD34/ABCG2,vWF/ABCG2, CD34/VEGF,VEGF/CD34 in glioma vessels,but no co-expression cells of GFAP/ABCG2.And co-expression cells of CD34/ABCG2,VEGF/CD34 were also observed in normal brain tissue,but no co-expression cells of ABCG2/GFAP,either.Conclusions:The co-expression of stem cells marker ABCG2 and vascular endothelia cells marker CD34 and vWF,but no co-expression ABCG2 and GFAP, indicated that glioma stem cells have ability to differentiate into glioma vascular endothelial cells for the co-expression of CD34/VEGF were also observed.The differentiation is also related with cell secretion of VEGF,but not with GFAP. PartⅢParticipation of cells coexpressing ABCG2 and nestin in glioma angiopoiesisObject:It is known that stem cells marker nestin and CD133 have close relationship in glioma stem cells.This article aims to investigate the relationship between nestin and another stem cells marker ABCG2,and to investigate the participation of cells coexpressing the two markers in glioma angiopoiesis.Methods:Glioma specimens with most plentiful vessels and highest pathological grades selected from 90 human glioma were immunostained against nestin and ABCG2, then observed with LSCM.Results:Some cells composing glioma vessels co-expressed nestin and ABCG2, and the heterogeneity among vessels were also observed.Nestin was expressed not only in the glioma vascular endothelial cells but also in tumor cells away from vessels.There were no co-expression of nestin and GFAP in glioma vessels.Conclusions:The glioma stem cells not noly have the potentiality to differentiate into their parent tumor cells but also into cells with phenotype of tumor vessel cells, which provide the tumor vessel formation theory:vasculogenic mimicry and mosaic vessels with new evidence.PartⅣAs a high risk factor,ABCG2 could effect survival of glioma patientsObject:While ABCG2 was a key gene for glioma angiogenesis,as discussed in first part and third part,it is worthy to make clear whether ABCG2 were associated with patient prognosis,which is the object of this part.Methods:A tissue microarray that include all pathological grades of human brain glioma tissues was constructed,and the expressions of PCNA,P53,VEGF,IGFBP-2, C-MYC and ABCG-2 were detected by EnVision immunohistochemical staining method in the tissue microarray.Cox proportional hazards models were usedunivariable analyses,and multivariabte analyses were conducted to characterize the association between survival and both clinical and biological markers.Results:Univariable analyses show that histopathology grade、ABCG2、P53 and VEGF was correlated with the prognosis(P＜0.05).ABCG2、P53 and VEGF immunopositivity enter the final multivariable model,and their high expression show poor prognosis.Conclusions:This study demonstrates ABCG2 being a high risk factor in glioma patients.This provides further evidence for target molecule theraty.更多还原