摘要
胶质瘤是人类最常见的难治性原发性颅内肿瘤,其发病机理、有效治疗方法仍在探索之中。近年分子生物学和分子遗传学在胶质瘤发病机理研究中的应用已取得初步进展,认识到胶质瘤的发生和其他肿瘤同样是涉及细胞周期调控、发育与分化、信号传导、凋亡与死亡和细胞代谢等等多种机制异常的病理过程。但目前仍不清楚促使胶质瘤的具体形成因素以及不同类型胶质瘤的分子病理的差异。而且,在不同组织中相同的基因及基因网络可能对胶质瘤的发生和发展具有不同的作用,研究胶质瘤中基因通路的异常及相同基因通路在不同来源组织之间的相互作用可能比研究单基因表达异常更能提供疾病发生、发展的生物学信息。因此,寻找与胶质瘤发病相关的基因,深入了解胶质瘤的分子病理,在此基础上开拓胶质瘤治疗的新策略和靶点,成为胶质瘤研究领域的一项重要内容。
我们前期应用Atlas cDNA微阵列方法比较研究了63例不同类型胶质瘤及5例正常脑组织的基因表达谱,结果发现与胚胎发育相关Notch信号通路的某些成员如Notch1在多数胶质瘤中均有差异表达,同时还发现应用RNA干涉技术敲低人脑胶质细胞瘤细胞系U251的Notch1表达后,细胞的增殖,侵袭受到抑制,凋亡增加。裸鼠体内移植瘤试验也证明敲低Notch1可以使肿瘤生长速度减慢,荷瘤动物生存期增加；说明Notch1具有促进胶质瘤生长的作用。但国外有报道显示,多见于小脑生长的髓母细胞瘤中, Notch1却起到抑制肿瘤的作用,而Notch家族的另外一个成员Notch2促进肿瘤生长,二者呈现相反的作用趋势。因此,本研究从大样本胶质瘤中的Notch基因表达着手,研究Notch家族成员在不同组织中的表达水平,筛选关键责任基因,然后以Notch2构建体转染胶质瘤细胞系进行体外实验,观察肿瘤细胞恶性表型变化；然后利用裸鼠荷瘤模型进行了Notch2构建体治疗胶质瘤的动物实验研究,以期进一步了解Notch通路在胶质瘤中的作用以及对胶质瘤恶性进展的影响,并初步研究了Notch信号通路与胶质瘤干细胞的关系。
本课题第一部分应用组织芯片免疫组化方法研究了60例胶质瘤标本、10例髓母细胞瘤标本及6例正常脑组织、应用Western Blot方法及Real-time PCR方法研究了手术切除新鲜胶质瘤标本34例、髓母细胞瘤标本4例及正常脑组织标本2例的Notch通路部分成员Notch1/2的表达及其与胶质瘤恶性程度之间的相关性。并以PCNA为增殖活性指标,以cyclin D1为细胞周期运行的指标,应用免疫组化法研究了Notch1/2、PCNA和cyclinD1的表达及其相关性。在大样本胶质瘤中Notchl/2的表达和分析发现Notch1在大脑生长的星形细胞瘤中高表达,并且表达水平随肿瘤恶性程度增加而明显上升,而Notch2无表达,而在小脑生长的髓母细胞瘤中,Notch2高表达,Notchl无表达。二者形成相反的趋势。对PCNA和cyclinD1的免疫组化分析发现：上述指标在胶质瘤中的表达阳性率和表达强度随肿瘤恶性程度增加而增加,在低级别和恶性胶质瘤之间存在显著性统计学差异；此外,相关分析发现Notchl/2、PCNA和cyclinD1之间存在着显著相关性。这些研究结果表明：Notchl通路对胶质瘤的恶性进展作用显著,激活这条通路可以促进肿瘤细胞增殖；而Notch2通路在大脑发生的星形细胞瘤中则起到明显的抑制作用。为进一步研究Notch2对胶质瘤生成的影响,构建了Notch2的真核表达载体。
第二部分将Notch2构建体-脂质体复合物直接转染人脑胶质瘤细胞系A172及U251, pcDNA3载体转染组为空载对照。G418筛选阳性克隆并应用RT-PCR、免疫荧光和蛋白印迹法鉴定转染成功。MTT法和Annexin V染色法评价细胞的增殖活性和凋亡,Transwell试验分析侵袭生长能力,流式细胞法分析细胞周期,以确定Notch2构建体对细胞生长的作用,并应用蛋白印迹分析转入Notch2构建体表达后该信号通路部分成员以及相关通路活性的变化,探讨可能存在的机制。体外试验发现,与对照组和转染空载pcDNA3组比较,转染Notch2构建体胶质瘤细胞增殖、侵袭能力受到抑制,并可诱发细胞凋亡,细胞周期分析出现SPF下降,G0/G1阻滞。同时发现：EGFR、p-AKT、PCNA、Bcl-2、MMP-9及CyclinD1几个重要癌蛋白的表达水平均有明显的下调,同时检测到肿瘤干细胞标记CD 133,Nestin表达下调,提示可能与肿瘤干细胞的生长抑制有关。
第三部分为体内实验,将Notch2 f低表达的U251细胞采用细胞悬液接种法和瘤组织块接种法接种于裸鼠右腿与腹腔邻接部位皮下建立U251细胞来源的胶质瘤移植瘤模型。以脂质体为介导给予Notch2构建体治疗,将裸鼠分为治疗组、空载组、对照组,每组8只。另取U251细胞来源的荷瘤鼠,给予靶向Notch1的RNAi干涉片段,分为RNAi干涉组、无义序列组、对照组,与Notch2构建体联合治疗组。动态测各组实验动物皮下肿瘤生长情况,观察28天取出肿瘤并应用免疫组化法检测肿瘤标本Notch1, Notch2, Bcl-2、Caspase-3、MMP2、MMP9, PCNA, Cyclin D1,AKT,p53,Western blot法检测CD133,Nestin并探讨Notch通路与肿瘤发展的关系。体内试验发现与对照组和转染空载pcDNA3组比较,转染Notch2构建体组后肿瘤生长明显减慢,肿瘤体积显著为小。免疫组化研究发现,转染Notch2构建体组较与对照组和转染空载pcDNA3组PCNA、MMP9、cyclin D1、Bcl-2、AKT、p-AKT的表达均下降,Caspase-3、p53表达上调,Western blot检测肿瘤干细胞标记CD133,Nestin下调。转染Notch2构建体组可见大量凋亡细胞,而对照组和无义序列组几乎没有凋亡细胞。Notch2+ Notchl siRNA组与Notch2,Notchl siRNA的单独转染组无明显差异,提示二者可能并无协同作用。
本课题第四部分构建与Notch通路上转录因子RBP-J结合序列的重组慢病毒表达系统,转染胶质瘤细胞系,通过流式细胞术筛选Notch通路激活的细胞群,探讨Notch信号通路与肿瘤干细胞关系。将与RBP-J结合序列RBS与EGFP序列插入质粒载体,构建入门质粒pRBS-EGFP,入门质粒与慢病毒载体重组,构建慢病毒表达载体pLenti-RBS-EGFP,转染包装细胞293T,包装产生慢病毒Lenti-RBS-EGFP。将慢病毒Lenti-RBS-EGFP转染胶质母细胞瘤细胞系KK,流式细胞仪分选EGFP+,EGFP-细胞,分析Notch通路激活细胞与未激活细胞群肿瘤干细胞标记的表达情况以及成瘤能力的变化。结果证明,Notch通路激活的细胞组分相对于未激活的细胞组分肿瘤干细胞的表面标记CD133,Nestin升高。反映细胞增殖的细胞因子Ki67升高,反映凋亡的细胞因子Caspase-3降低。克隆形成试验显示Notch通路激活组细胞有更强的成瘤能力。通过慢病毒介导的RBP-J结合序列,能特异地反映Notch通路的活性。基于Notch通路的激活分选出的细胞组分,具有肿瘤干细胞的特性,并且有极强的成瘤能力。
本研究表明：Notch通路在胶质瘤中异常激活,故在胶质瘤发生、发展过程中具有其重要性。基于不同组织来源的肿瘤,Notch家族成员表达不同,可能具有不同的作用。与髓母细胞瘤中相反,在大脑胶质瘤中Notchl表达增高,Notch2表达低下；我们将Notch2构建体转染人胶质瘤细胞系,可见细胞SPF降低,G0/G1期出现阻滞,并抑制细胞侵袭、增殖,促进凋亡。脂质体介导Notch2体内治疗可显著抑制肿瘤生长,促进细胞凋亡。抑制侵袭。由此,可初步认定Notch2通路在大脑发生的星形细胞瘤中具有抑制肿瘤的作用,并可成为胶质瘤基因治疗的候选基因。Notch与肿瘤干细胞关系密切,值得进一步研究。
Glioma is the most common and incurable intracranial tumor. Its pathogenesis and effective therapeutic modalities still have to be studied. As the knowledge of tumor biology and molecular genetics increased, it has been shown that the development of gliomas, just like the tumors in other sites of the body, is involved in the processes of uncontrolled cell proliferation, cell dedifferentiation and dysregulation of cell apoptosis. However, up to date, it is still not clear which are the initiating molecular events in detail. In addition, gene products exert their function through signaling pathways and there are links and crosstalks even forming a complex network among different signaling pathways in different tissues. Accordingly, it is more important to study the abnomal activities of gene transduction pathways or the crosstalks among the different gene transduction pathways than to study a single gene activity. To optimize treatment strategies and to develop novel therapeutic approaches for gliomas, a more precise understanding of the cellular and molecular basis of gliomas is necessary, so it is important to continue seeking more genes which may play a key role in the gliomagenesis.
In our previous work, the mRNA expression profiles of 63 samples of different pathological types of human gliomas as well as 5 samples of human normal brain tissues were selectively analyzed by Atlas Human Cancer Array 1.2. It had been observed that some members of Notch pathway were differentially expressed. Meanwhile, we also found inhibition of proliferation, cell differentiation and induction of cell apoptosis when the Notch 1 expression was knocked down by RNAi technology. It also had been demonstrated that the growth of xenograft tumors in nude mice step down. This evidence indicated that overexpression of Notch 1 can cause glioma. But, according to some reports, elevated expression of Notch2 was detected in contrast with negative expression of Notchl in medulloblastomas. Notchl and Notch2 have different expression in astrocytomas and medulloblastomas. For further studying the aberration and the role of Notchl and Notch2 in gliomas, we began with examining the expression of Notchl,2 in a large number of glioma specimens in the present study and aimed at better understanding of the expression of the major genes in the Notch signaling pathway and interruption of Notch pathway in the prevention of progression of gliomas. Moreover, the established subcutaneous xenograft gliomas in nude mice were treated with Notch2 cDNA for further defining the role of Notch2 in the progression of gliomas.
The present study was divided into four parts.
The first part of this study focused on the expression of Notch 1/2 in 60 samples of gliomas and 10 samples of medulloblastomas,6 samples of normal brain tissues in tissue array and their correlation with degree of malignancy of gliomas by immunohistochemcal staining. In addition, freshly resected samples including 34 samples of gliomas with different grades,4 samples of medulloblastomas and 2 samples of normal brain tissues were examined by Western Blotting and Realtime PCR. Immunohistochemcal staining was also used for PCNA, and cyclinD1 expression and their correlation was analyzed. Based on these findings, a eukaryotic expression vector of Notch2 was constructed for studying its biological effect on glioma cell growth. Notch 1 mRNA expression increased correspondingly to the ascending order of tumor grade but Notch2 mRNA expression can hardly be detected by Realtime PCR in gliomas. In immunohistochemical study, the protein expression of Notchl, PCNA and cyclinDl also increased with the degree of malignancy of tumors, and there was statistically significant difference between their expression in low-grade and high-grade tumors. In addition, the expression of Notchl, PCNA, and cyclinDl was correlated positively with each other. Such results imply that aberrant Notch pathways might be important for the progression of malignant gliomas, either overexpression of Notchl or depletion of Notch2 may contribute to tumor proliferation and invasion, which are the important phenotypes of malignant gliomas.
In the second part of this study, Notch2 construct was transfected to U251 and A172 malignant glioma cells. The positive clones were selected by G418 and identified by RT-PCR, immunofluorence and Western blot analysis. For observing the phenotypic changes of the cells transfected with Notch2, The cell proliferation was determined by MTT assay and cell cycle was detected by flowcytometry, cell apoptosis was detecteded with Annexin V staining and cell invasion was evaluated by Transwell test. Moreover, the molecules regulating the cell proliferation, invasion and apoptosis were examined by immunofluorence staining and Western blot analysis. The major members of PI3K/AKT pathway were also detected by Western blotting so as to explore the link or crosstalk between Notch and AKT pathways. Meanwhile, cancer stem cell marker CD 133 and Nestin also had been detected to show the relationship between Notch signaling and cancer stem cells. As compared to control and empty vector transfected cells group, the proliferation and invasion activity were inhibited and apoptotic cells increased in U251 cells transfected with Notch2 constructs, the cell cycle analysis showed the lowering SPF and arrest of cells in G0/G1 phase. At the same time, the expression of p-AKT was downregulated and cancer stem cell marker expression including CD 133 and Nestin also decreased.
In the third part of the present study, in vivo experiment was carried out. Nude mice with well-established subcutaneous explanted U251 gliomas were divided into six groups:1) control group:gliomas treated with PBS; 2) empty vecter group; gliomas treated with empty vectors; 3) Notch2 group:gliomas treated with Notch2-lipofectamine complex; 4) nonsense siRNA group; 5) Notchl siRNA group: gliomas treated with Notchl siRNA; 6) Notch2+Notchl siRNA group:gliomas treated with Notch2 plasmid and Notchl siRNA synchronously. Each group consists of eight nude mice. Tumor volume was measured regularly in subcutaneous glioma models. Notchl Notch2、Bcl-2、Caspase-3、MMP2、MMP9、PCNA、Cyclin D1、AKT、p-AKT、p53 were detected by immunohistochemistry, CD133、Nestin were detected by Western blotting in tumors of each group. Apoptosis was also detected with TUNEL method. Compared to control and empty vecter group, the tumors in mice treated with Notch2 constructs grew slowly and the tumor volumes were much smaller than those in control group. Also, as compared to control and nonsense siRNA treated group, the tumors in mice treated with siRNA targeting Notchl grew slowly and the rumor volumes were much smaller than those in control group. By immunohistochemical and Western blotting study, the expression of Bcl-2、MMP2、MMP9、PCNA、Cyclin D1、AKT、p-AKT、CD133、Nestin of tumors treated with Notchl siRNA and Notch2 constructs were decreased. A lot of apoptotic cells could be found in tumors with treatment of Notchl siRNA and Notch2 construct group, only a little apoptotic cells were found in control and nonsense transfected group as well. But combinations of these two treatments are not more efficient than using them singly. It maybe suggested that there is not synergetic and superposable between these two treatments.
The fourth part included construction of Lentiviral expression vector of RBP-J banding sequence in Notch signaling pathway, Lentiviral construct infecting to glioma cell line, sorting out Notch activity cell population by flow cytometry and studying the relationship between Notch signaling and tumor stem cells. Entry plasmid pRBS-EGFP was constructed by insert the RBP-J banding sequence RBS and EGFP to plasmid vector.The entry plasmids and lentiviral vector were recombinated and lentiviral expression plasmids pLenti-RBS-EGFP was constructed. Lentiviral expression plasmids and packaging plasmids were transfect to 293T cells to produce Lenti-RBS-EGFP virus. Glioblastoma cell line was infected by Lenti-RBS-EGFP and flow cytometry was used to sort the EGFP+, EGFP-cells. According to the result, the cell population with Notch activity expressed higher tumor stem cells marker CD 133 and Nestin as compared to cell population without Notch activity. Cell proliferation marker Ki67 was increased and apoptosis factor caspase-3 was reduced. Colony-forming assays suggest that cell population with active Notch signaling posesses a stronger ability of tumorigenisis.
From the present study, it has been demonstrated that Notch 1 was overexpressed and Notch2 was deleted in gliomas. Transfection of Notch2 gene reversed the malignant phenotypes of glioma cells while inhibition of Notch 1 gene expression by RNAi technology had the similar effect. So Notch signaling pathway may play an important and complex role in the gliomagenesis and Notch can be the candidate for gene therapy of human gliomas. The relationship between Notch and cancer stem cell should be further investigated.
引文
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