【作者】 赵振宇；

【导师】 卢亦成； 陈菊祥； 胡国汉； 骆纯； 孙克华；

【作者基本信息】 第二军医大学 ， 外科学， 2010， 博士

【摘要】 胶质母细胞瘤(GBM)是脑肿瘤中最常见的一种,约占胶质瘤的69%。因为治疗效果不理想,生存时间常少于1年。由于胶质瘤呈浸润性生长,有丰富的新生血管,手术治疗很难完全清除肿瘤细胞,因此化疗是GBM患者辅助治疗的重要手段之一。它可以进一步杀灭残留的肿瘤细胞,尤其对肿瘤部分切除患者更有益处。但现阶段GBM患者化疗后生存时间的延长和生活质量的提高程度并不令人满意,胶质瘤细胞对化疗药物耐受是导致有效率低的重要原因。相同年纪、KPS的GBM患者接受相同的治疗预后差异却很大,说明肿瘤患者自身存在不同耐药的分子机制。基因芯片以其高通量、自动化、简便易执行等优势成为研究肿瘤分子学机制的重要技术,我科前期已制成3批基因芯片,但研究结果产生的大量数据往往是进行深入实验的最大困难。本实验旨在应用生物信息学技术,利用相关数据库资源,通过计算机分析、统计学判别,结合患者完整的生存资料,对于芯片产生的大量数据进行系统地挖掘和分析,筛选出影响GBM生存时间和化疗敏感性的相关基因。通过生物信息学分析,筛选得到29个核心基因与GBM患者生存时间和化疗敏感性密切相关。其中分化抑制因子2(inhibitor of differentiation 2/inhibitor of DNA binding 2, Id2)因在脑组织及其他肿瘤中广泛表达,成为接下来实验关注的对象。Id2是一类在细胞分化、增殖过程中广泛表达的转录调控因子,属于螺旋-环-螺旋(helix-loop-helix, HLH)家族的分化抑制因子Id蛋白家族成员。Id2具有调控细胞周期、细胞增殖、促进胚胎形成和器官形成、抑制细胞分化、诱导细胞凋亡、维持细胞存活、促进血管形成及肿瘤浸润、发挥癌蛋白功能等生物学作用。目前,关于Id2在人脑胶质细胞瘤组织中表达水平及与人脑胶质瘤的生物学行为相关性的研究尚不深入。本研究拟通过检测Id2在不同病理级别脑胶质瘤以及恶性胶质瘤细胞株中的表达情况,构建Id2基因RNA干扰慢病毒载体,并通过siRNA干扰手段在人GBM细胞株中沉默Id2,检测其对Me-CCNU, VM26, TMZ等胶质瘤常用化疗药物敏感性的影响,为临床治疗脑胶质瘤制定个体化治疗方案、明确新的分子靶标提供依据。实验共分为四个部分：第一部分,应用生物信息学筛选人胶质母细胞瘤化疗相关的基因；第二部分,Id2在人脑胶质细胞瘤组织中的表达研究；第三部分Id2基因RNA干扰慢病毒载体的构建和鉴定；第四部分,siRNA靶向性抑制Id2表达对胶质母细胞瘤化疗敏感性的影响。第一部分应用生物信息学筛选人胶质母细胞瘤化疗相关的基因目的：本部分实验旨在应用生物信息学技术筛选影响胶质母细胞瘤患者生存时间和化疗药物作用相关的基因。方法：对2批胶质瘤患者Biostar基因芯片和1批胶质瘤患者Affymetrix基因芯片进行分析。通过长期随访,完善芯片中胶质瘤患者的临床资料。以生存时间12个月将胶质母细胞瘤患者分为生存时间长、短两组,筛选两组间差异基因及与化疗药物作用相关的差异基因,明确差异基因参与的功能和通路,并构建与化疗药物作用相关基因参与的信号传导网络,从而筛选出影响化疗效果的核心基因。结果：2003年BIOSTAR芯片,2007年BIOSTAR芯片和2007Affymetrix芯片中GBM生存时间长短两组间与化疗药物相关的差异基因分别是429条,74条,2018条。3批芯片的差异基因主要参与81项基因功能,主要参与36条信号传导通路。通过对差异基因功能、通路、网络的分析,得到影响胶质母细胞瘤化疗药物作用的核心基因29条。结论：通过严谨的实验设计和科学的统计学判别,结合患者完整的生存资料,可应用生物信息学对基因芯片的大量数据进行挖掘和分析。筛选出29条影响GBM患者生存时间,影响GBM化疗药物作用的核心基因,为进一步功能实验奠定基础,为GBM患者的预后判断和个性化治疗提供依据。第二部分Id2在脑胶质瘤组织和胶质瘤细胞株中的表达研究目的：研究Id2在正常脑组织、不同级别脑胶质瘤组织及胶质细胞株和恶性胶质瘤细胞株U87、U251、SHG-44中的mRNA和蛋白表达水平。方法：应用real-time PCR检测Id2 mRNA的在28例不同级别脑星形胶质细胞瘤和4例正常脑组织以及胶质细胞株和恶性胶质瘤细胞株U87、U251、SHG-44中的表达。应用免疫组织化学染色检测Id2在56例脑星形胶质细胞瘤组织和4例正常脑组织中的蛋白表达水平,应用Western blot检测Id2的在28例不同级别脑星形胶质细胞瘤和4例正常脑组织的蛋白表达水平。结果：real-time PCR结果提示Id2 mRNA表达水平在星形胶质瘤组织中明显上调,与正常脑组织相比差异有统计学意义(43.53±35.32 vs 1.89±1.16；P=0.000)。Id2 mRNA水平在高级别胶质瘤组织中的表达水平明显高于低级别胶质瘤,二者之间差异有统计学意义(59.71±43.82vs10.24±8.46；P=0.000)。免疫组织化学染色提示Id2蛋白在星形胶质细胞瘤组织中表达明显上调,与正常脑组织相比差异有统计学意义(7.90±5.70% vs 0.0±0.0%；P=0.000),且高、低级别胶质瘤之间差异有统计学意义(10.53±5.79% vs 4.23±2.85%；P=0.000)。Western blot结果提示Id2在星形胶质细胞瘤中的表达高于正常脑组织(0.61±0.48 vs 0.09±0.07；P<0.01),且高、低级别胶质瘤之间的Id2表达有明显差异(1.17±0.84 vs 0.33±0.18；P<0.05)。Id2在人胶质母细胞瘤细胞株U87、U251、SHG44中的表达高于正常胶质细胞株(1.25±0.89 vs 0.49±0.35,P<0.05)。结论：Id2在星形胶质细胞瘤组织中的mRNA和蛋白表达水平明显高于正常脑组织,提示Id2可能在星形胶质瘤的发生、发展过程中起重要作用。第三部分Id2基因RNA干扰慢病毒载体的构建和鉴定目的：构建针对人Id2基因的短发夹状小干扰RNA(shRNA)表达载体。方法：设计并合成4对Id2靶向的发夹状siRNA靶序列,合成靶序列的OligoDNA,退火形成双链DNA,与经AgeI和EcoRI酶切后的pGCSIL-GFP载体连接产生短发卡RNA慢病毒载体,PCR筛选阳性克隆,测序鉴定。结果：PCR鉴定与DNA测序证实合成的含Id2 shRNA慢病毒载体寡核苷酸链插入正确。结论：成功构建高效阻断Id2基因表达的RNAi慢病毒表达载体,为应用RNAi进一步研究Id2基因的对胶质瘤化疗敏感性的影响奠定基础。第四部分siRNA靶向性抑制Id2表达对胶质母细胞瘤化疗敏感性的影响目的：研究靶向Id2的siRNA干扰对U87胶质瘤细胞株对Me-CCNU、VM26、TMZ敏感性的作用。方法：将鉴定有效的靶向Id2的siRNA的慢病毒载体感染U87细胞株,Western Blot法检测转染后蛋白表达水平。MTT法测定感染后36小时Me-CCNU、VM26、TMZ培养U87细胞增殖率。结果：Id2 siRNA可有效沉默U87细胞Id2的表达。MTT法检测Id2 siRNA组在Me-CCNU、VM26、TMZ三种化疗药物中36h的细胞生长抑制率分别是39.75%±3.62%,40.6%±7%,20.03%±2.56%；较空质粒转染组(22.24%±6.77%,16.73%±5.44%,8.4%±2.26%)增高,差异均有统计学意义(P<0.05)。结论：通过RNA干扰沉默Id2在U87胶质瘤细胞中的表达,可有效提高肿瘤细胞对Me-CCNU、VM26、TMZ的化疗敏感性。抑制Id2表达可以提高恶性胶质瘤细胞对于化疗药物的敏感性,可能成为恶性胶质瘤化疗方案制定的依据和靶向治疗的选择。更多还原

【Abstract】 Glioblastoma (GBM) is the most common malignant tumor of the central nervous system and accounts for approximately 45～55% of gliomas. The mean survival of GBM is less than one year. As GBM is highly invasive and prone to angiogenesis, complete surgical resection is difficult. Chemotherapy is therefore an important adjuvant treatment for GBM patients by killing residual tumor cells and prolonging the survival duration of patients who have undergone partial tumor resection in particular. GBM is not satisfactory for improve in survival time and quality of life after chemotherapy. Resistance to chemotherapeutic drugs is an important reason for low efficiency. The survival duration of GBM patients with similar ages and KPS scores varies greatly after the same treatment. The reason may be due to different tumor moleculogy in these patients.Gene microarrays were used to study tumor and drug activity-related genes,3 glioma gene chips have been made in our lab, the greatest difficulty is how to make scientific analyses of the huge amounts of complex data obtained and determine the main responsible genes. The present study made use of bioinformatics method to analyze the data obtained from microarrays in combination with the complete data about survival of GBM patients in an attempt to investigate CCNU sensitivity-related genes in GBM patients. We believe that using these genes to assess the prognosis of GBM patients would be more accurate than conventional histopathology, and more helpful in working out more specific and individualized targeted treatment regimens according to genetic characteristics of individual patients.Through the bioinformatics analysis,29 core genes related to glioblastoma survival time and chemotherapy sensitivity were screened out. Among of them, inhibitors of differentiation 2 (Id2) become the object to next test. Id2 is a member of family of expressed transcription factor of cell differentiation and proliferation, belonging to helix-loop-helix (HLH) protein family members. Id2 has been demonstrated to control cell cycle, cell proliferation, to promote embryo and organ formation, to inhibit cell differentiation, induce apoptosis, to maintain cell survival and promote blood vessel formation, and play an important biological role in tumorigenesis. At present, research on the Id2 in glioma tumorigenesis and biological behavior was few reported.This study was designed to detect the expression of Id2 in various pathological grade brain gliomas and malignant glioma cell lines, to construct Id2 gene RNA interference lentiviral vector. To knock-down Id2 through siRNA interference in U87 cell lines, change of sensitivity to chemotherapeutic drugs Me-CCNU, VM26, TMZ is to be detected. The research would show great promise of individualized treatment programs and a new molecular target to GBM. This study is divided into four parts: the first part is to use bioinformatics method to investigate the genes related to chemosensitivity in human glioblastoma, the second part is to investigate the expression of Id2 in astrocytic tumors and glioma cell lines, the the third part is to construct and identificate lentiviral vector of RNAi of Id2 gene, and last part is to knock-down expression of Id2 in U87 cell line by lentivirus-mediated RNAi and to observe its effect on Me-CCNU, VM26, TMZ chemosensitivity.PartⅠUsing Bioinformatics Method to Investigate the Genes related to Chemosensitivity in Human GlioblastomaObjective:In this part of study, bioinformatics method was used to analyze data of cDNA microarray to investigate the genes related to survival time and drug sensitivity in GBM.Methods:Biostar and Affymetrix Microarray of GBM patients were analyzed, and clinical data of these patients in the microarray were perfected through long-term follow-up study. According to survival time of 12 months, the samples were divided into two groups. Differential expression genes between the long- and short- survival groups were picked out, GO-analysis and pathway-analysis of the differential expression genes were performed. Drug-related signal transduction networks were constructed. The methods combined three steps before were used to screen core genes that influenced chemosensitivity.Results:In 2003 Biostar microarray,2007 Biostar microarray and 2007 Affymetrix microarrays, there were 429,74,2018 differentiate genes that influenced survival duration of GBM respectively. They mainly participated in 108 gene functions and 94 signaling transduction pathways, based on which 29 core genes that influenced chemosensitivity of GBM to Chemistry drug were obtained.Conclusion:With complete clinical information, mass data of cDNA microaray can be further analyzed by using bioinformatics method.29 genes were found to predict the clinical outcomes and have a significant influence on chemosensitivity in GBM. This study will provide a foundation not only for the further functional research but also for prediction of prognosis and fulfillment of personalization chemotherapy in GBM.PartⅡExpression of Id2 in Astrocytic Tumors and Glioma Cell LinesObjective:To investigate expression level of Id2 mRNA and protein in normal brain tissues, astrocytomas of different grades and U87, U251 and SHG-44 Glioma Cell Lines.Methods:The expression levels of Id2 mRNA were evaluated by real-time quantitative PCR in 28 astrocytic tumors,4 normal brain tissue samples and U87, U251 and SHG-44 Glioma Cell Lines. By using immunohistochemistry, expression levels of Id2 protein were assessed in 56 astrocytic tumors of different pathological grades and 4 normal brain controls. By using Western blot, expression levels of Id2 protein were assessed in 28 astrocytic tumors of different pathological grades and 4 normal brain controls.Results:Quantitative real time PCR analysis demonstrated elevated expression levels of Id2/β-actin in high-grade astrocytomas versus low-grade (59.71±43.82vs10.24±8.46; P=0.000) and gliomas vs normal brain tissues(43.53±35.32 vs 1.89±1.16; P=0.000). It also demonstrated elevated expression levels of Id2/β-actin in U87, U251 and SHG-44 glioma cells versus astrocyte cell line(33.77±21.54,30.20±20.13 vs 1.00±1.57;P=0.000). Further, Id2 immunoreactivity was predominantly detected in the cytoplasm of tumor cells, whereas no positive staining for Id2 was observed in normal brain tissues. Statistical analysis showed Id2 increase in high-grade astrocytomas versus low-grade tumors (10.53±5.79% vs 4.232.85%; P=0.000) and gliomas vs normal controls (7.90±5.70% vs 0.0±0.0%; P=0.000). Western blot analysis showed elevated expression levels of Id2 in high-grade astrocytomas versus low-grade (1.17±0.84 vs 0.33±0.18; P<0.05) and gliomas vs normal brain tissues (0.61±0.48 vs 0.09±0.07; P<0.01), and elevated protein expression levels of Id2 in U87, U251 and SHG-44 glioma cells versus astrocyte cell line(1.25±0.89vs 0.49±0.35, P<0.05).Conclusion:Id2 is present in different grade gliomas and its expression levels significantly increase at both mRNA and protein versus normal brain tissues. There existed significant difference in both mRNA and protein leves of Id2 between high-grade astrocytomas and low-grade. Id2 is also present in glioma cell lines and its expression levels significantly increase at both mRNA and protein levels versus astrocyte cell line. These data suggest that overexpression of Id2 may serve as one important molecular mechanism that underlies the development and progression of astrocytic tumors.PartⅢConstruction and Identification of Lentiviral Vector of RNA Interference of Id2 GeneObjective:To construct a lentiviral vector of RNA interference(RNAi)of human Id2 gene.Methods:Four shRNA sequences targeting human Id2 gene were designed and then the complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, to construct a lentivirus vector which expressed short hairpin RNA (shRNA), and it was identified by PCR and DNA sequencing.Results:PCR identification and DNA sequencing demonstrated that insertion of oligonucleotide of the lentivirus RNAi vector containing human Id2 shRNA was effective.Conclusion:The high efficient lentivirus RNAi vector of Id2 was constructed successfully as the basis for further study of its influence on chemosensitivity in GBM.PartⅣSuppression of Brain Glioma Cells Expression of Id2 by Lentivirus-mediated RNAi and its Effect on Me-CCNU, VM26, TMZ ChemosensitivityObjective:To investigate the Me-CCNU, VM26, TMZ chemosensitivity change in U87 glioma cell after Id2 inhibition by lentivirus-mediated RNA interference. Methods:Lentiviral vectors of Id2 siRNA was transfected into brain glioma cell line U87, the expression of Id2 was determined by western blot. The effects of drugs on proliferation of RNAi, negative and control cells were assessed by MTT assay.Results:The expression level of Id2 protein was knocked down by Id2 siRNA as indicated by western blotting analysis. MTT assay showed the inhibition rate of U87 glioma cells cultured in Me-CCNU, VM26, TMZ was significantly higher in Id2-shRNA group (39.75%±3.62%,40.6%±7%,20.03%±2.56%) compared with that of vector control (22.24%±6.77%,16.73%±5.44%,8.4%±2.26%, p<0.05).Conclusion:RNAi vectors of Id2 could effectively suppress its exprssion in U87 cell line, and enhance the sensitivity of glioma cells to Me-CCNU, VM26 and TMZ. Given the great impact on sensitivity to chemotherapeutics drugs of GBM, knock-down of Id2 expression may therefore represent a potentially individualized treatment strategy against GBM.更多还原