Nogo-B在人胶质瘤组织和微血管内皮细胞中差异表达的研究
摘要
颅内绝大多数肿瘤都起源于星型细胞、少突胶质细胞、许旺氏细胞或其前体细胞,它们是神经元的支持细胞,这些肿瘤中以星型细胞瘤最为常见,占中枢神经系统肿瘤的40%以上。WHO按组织病理学将星型细胞瘤分为4级:Ⅰ级和Ⅱ级星型细胞瘤为低度恶性,而Ⅲ级和Ⅳ级为高度恶性。Ⅲ级胶质瘤很容易发展为Ⅳ级星型细胞瘤,即多型胶质母细胞瘤。
胶质瘤新生血管的形成在肿瘤发生发展过程中起着重要作用,低度恶性胶质瘤低到中度血管化,而高度恶性的胶质瘤具有较高的血管密度。我们知道,肿瘤扩张必须通过血管生成,即“血管化转换”(Angiogenic switch,AG),以获取氧气和营养,AG是通过肿瘤表达促血管生成因子(包括多肽和蛋白)而完成的,促血管生成因子的生成与环境信号和遗传因子有关,主要包括缺氧、低PH值、低血糖、肿瘤内压力增加、免疫系统所诱发的免疫反应、基因突变等等。
研究表明,胶质瘤血管在结构和功能上都与正常血管不同。例如,肿瘤血管缺乏保护性的生长调节机制,并缺乏有功能的组织特异性血管周细胞。此外,血管并不总是由同源性的内皮细胞层所构成,有时肿瘤细胞和内皮细胞混杂在一起,肿瘤血管管腔差别较大,通透性较高。胶质瘤表现为高度血管化,并且微血管与正常血脑屏障不同,血管通透性增高,由此可引起脑水肿。
此外,胶质瘤脑血管超微结构的研究发现微血管内皮之间紧密连接出现异常,紧密连接蛋白的缺失与血脑屏障的开启密切相关。另外,肿瘤血管形成需要不同的分子信号。例如内皮细胞的趋化和增生信号,细胞基质受体的表达,与细胞外基质降解相关的蛋白酶的表达等。
Nogo-B蛋白是一类中枢髓鞘源性抑制蛋白,是抑制中枢神经元轴突再生的抑制因子,分布广泛,在神经系统中高表达。在外周组织如血管组织、肾脏、肺脏、软骨、肾脏、皮肤、脾中也有表达。
组织损伤后Nogo-B的作用首先表现在参与中枢神经系统轴突再生抑制。Neuron杂志2003年发表了3篇有关Nogo基因缺陷小鼠文章。Strittmatter和同事发现Nogo-A/Nogo-B缺陷小鼠的轴突再生能力改善;Tessier-Lavigne团队分别制造出Nogo-A,B,C3亚型同时缺失模型和Nogo-A,B缺失模型,未发现轴突再生改善;Simonen等人报道伴随Nogo-A缺陷小鼠轴突生长抑制作用降低,中枢神经系统中Nogo-B表达上调。虽然以上文献报道有关Nogo-B功能略有矛盾,但是在某种程度上证明了Nogo-B参与中枢系统损伤后再生。
2004年Nature Medicine上的一篇文章充分揭示了Nogo-B限制外周血管损伤后的再生。Acavedo等人采用质谱分析和RT-PCR等技术首先发现血管的内皮细胞和平滑肌细胞中Nogo-B高表达,进而将其功能片段分别引入这两个细胞系,发现Nogo-B促进内皮细胞的迁移和抑制平滑肌细胞的再生,采用Nogo-B缺失小鼠研究发现,股动脉损伤后平滑肌细胞再生活跃造成血管闭塞,转染Nogo-B后平滑肌增生减弱。
国内Jing-Wei Pan等在2007年发表了Nogo-B和主动脉内皮细胞的关系的文章。文章报道在粥样硬化斑块组织中Nogo-B的mRNA和蛋白表达下调和动脉瘤的形成密切相关,Nogo-B在其中起保护血管的作用。在国外有研究建立了体外细胞模型,发现了Nogo-B更值得关注的功能,其可溶性N端片段对于内皮细胞具有显著的类似VEGF样趋化修复作用,但对于血管平滑肌细胞有阻断PDGF诱导的增生作用。该发现产生了巨大影响,Raines EW在Nature Medicine上作出“Nogo-B为血管病变安装了闸门”的评论,高度评价了Nogo-B在血管病变中的重要功能。
但是Nogo-B在血管病变损伤中的作用机制仍未能完全明确。英国Rousseau S等的意外发现表明,Nogo-B为SAPK2a/p38a通路中MAPKAP-K2的磷酸化底物,提示Nogo-B活性与MAPKs通路密切相关。MAPKs信号通路由一组以级联方式依次活化的丝/苏氨酸蛋白激酶组成,将与细胞膜受体结合的胞外刺激逐级放大并传导到细胞内,调节效应分子参与细胞生长、分化和凋亡等阶段的病理生理活动。其中P38和P42/44已被证明在内皮细胞的迁移和平滑肌细胞增殖中起重要作用。Nogo-B在MAPKs通路中的功能为Nogo-B的机制研究提供了线索,提示Nogo-B对血管的保护作用与MAPKs通路有关。
除了对调节血管生成的作用外,Nogo-B对肿瘤起了到底什么作用?有文献报道Nogo基因表达导致肿瘤细胞凋亡,这个基因在恶性肿瘤细胞系中有一个很强的促凋亡倾向,例如SaOS-2,HT-1080等。这些发现表明Nogo基因是一种新型促凋亡基因。研究表明这些凋亡蛋白位于内质网上,它编码内质网reticulum靶蛋白,其通路和目前已知的前凋亡蛋白不同,在肿瘤细胞中该基因的丢失非常明显。尽管研究表明Nogo-B基因导致凋亡,它不和目前已知的任何一种凋亡相关基因或者功能域同源。相反,它有独特的功能基序,即在它的C末端有一个重复赖氨酸内质保守基序,这个抗凋亡作用与该C-末端分子区域有关,但目前尚不清楚这一作用是否与NgR有关,Nogo-B可与内质网上促凋亡基因Bcl-x1、Bcl-2作用,诱导内质网张力改变和Ca~(2+)内流,影响肿瘤生长。
对于中枢神经系统肿瘤,特别是胶质瘤,目前只有Nogo-A方面的研究内容。Nogo-A的胞外功能域Nogo-66和髓鞘相关糖蛋白(Myelin-associated glycoprotein,MAG)可以让人胶质瘤细胞系U87MG的黏附性和迁移能力显著下降。他认为Nogo-66和MAG可以通过NgR激活,调节胶质瘤生长和迁移,这个现象具有潜在的临床治疗应用价值。但是目前尚没有Nogo-B和胶质瘤的报道。更多的是VEGF、PDGF等常见血管增殖相关生长因子方面的研究。
我们知道VEGF是一种与血管增殖相关生长因子,又称血管通透性因子。其主要生物学活性有:1、促进内皮细胞的增殖。2、增加血管的通透性、维持毛细血管周围内皮细胞的通透性。3、改变细胞外基质,促进血管生长,促进血管构建。4、促进血管形成前暂时性基质沉积。5、选择性激活小动脉、小静脉的内皮细胞,促进内皮细胞分裂、增殖。6、促进内皮细胞的趋化。7、保护新生血管。
已有文献报道恶性肿瘤中肿瘤细胞和血管内皮细胞中VEGF都有表达,且随着肿瘤恶性度的增加,VEGF的表达增加。VEGF主要分布于高度恶性的胶质瘤细胞中和血管内皮细胞,定位于细胞浆,低恶性胶质瘤表达相对较低,正常脑组织几乎检测不出。最新的研究表明肿瘤细胞表面也有VEGF受体高表达,并且受VEGF信号通路的调节,且很多肿瘤高表达VEGF,肿瘤细胞的生长和存活在部分程度上也受VEGF通路的调节。因此阻断VEGF的信号传导通路可以从多个方面抑制肿瘤的生长。目前已有报道采用VEGF单克隆抗体、VEGF受体拮抗剂等抑制肿瘤生长,最近还有利用RNA干扰技术,利用VEGF siRNA实行抗肿瘤血管生成疗法的报道,取得了一定进展。
RNA干扰(RNA interference,RNAi)是指细胞中导入与内源性mRNA编码区某段序列同源的双链RNA(dsRNA),可致该mRNA发生特异性降解从而导致基因表达沉默的现象。因基因沉默现象发生在转录后水平,又称为转录后基因沉默(PTGS)。目前,RNAi技术作为一个强有力的遗传学工具,提供了高效、特异性的抑制基因表达的手段,成为基因功能研究、抗病毒感染研究和基因治疗研究的重要方法。
恶性胶质瘤的发生、发展离不开微血管内皮细胞,微血管内皮细胞同样受到恶性胶质瘤细胞分泌的各种细胞因子的作用。目前对这些因子中的Nogo-B蛋白的研究主要集中于周围血管的内皮细胞上,在中枢神经系统中,Nogo-B对胶质瘤细胞和胶质瘤微血管内皮细胞到底起了什么样的作用,Nogo-B和VEGF之间到底存在什么样的关系,VEGF是否参与了Nogo-B的调节通路,如何利用Nogo-B和VEGF实行抗肿瘤血管生成疗法,这些是本课题的研究内容和目的。
研究分四个部分,第一部分:人胶质瘤组织中Nogo-B的功能表达。第二部分:人胶质瘤源性微血管内皮细胞中Nogo-B的功能表达。第三部分:VEGF参与胶质瘤微血管内皮细胞Nogo-B表达的调控机制。第四部分:干扰Nogo-B表达抑制微血管内皮增生。
研究采用以下研究方法:1、免疫组织化学、Western blotting法、Real-time PCR法分析在不同级别的胶质瘤组织中Nogo-B在蛋白和mRNA水平上的功能表达。2、利用免疫磁珠内皮细胞分选系统纯化胶质瘤源性微血管内皮细胞(Glioma derivedmicrovascular endothelial cells,GDMECs)。3、利用免疫组织化学法、Western blotting法、Real-time PCR法分析Nogo-B在不同级别的GDMECs中的功能表达。4、将VEGFsiRNA瞬时转染至GDMECs中,采用ELISA法、Real-time PCR法分析VEGF对GDMECs中Nogo-B表达的功能调控。5、采用小鼠脑血管内皮细胞系bEnd.3,将Nogo-B siRNA和bEnd.3共培养,观察bEnd.3增殖情况,探讨以Nogo-B作为靶点实行抗胶质瘤血管生成疗法的可行性。
本研究得到如下结果:1、人胶质瘤组织中Nogo-B与正常脑组织相比,染色阳性率降低。2、在高级别的胶质瘤中Nogo-B在蛋白和mRNA水平上表达下调,Nogo-B表达水平随着肿瘤恶性程度增加而显著降低。3、在GDMECs中,Nogo-B蛋白、mRNA表达强度随着其来源胶质瘤的恶性程度增加而显著增加。4、瞬时转染VEGFsiRNA到GDMECs后,Real-time PCR、ELISA结果表明干扰VEGF表达能显著促进降低Nogo-B的蛋白表达。5、Nogo-B siRNA和bEnd.3细胞共孵育后,bEnd.3中Nogo-B表达显著抑制,其抑制程度具有siRNA剂量依赖性;抑制Nogo-B表达后,和对照组相比,bEnd.3增殖受到抑制。
本研究得出结论:1、在人胶质瘤组织中,Nogo-B在蛋白和mRNA水平的表达与肿瘤恶性度成负相关。2、在GDMECs中,Nogo-B在蛋白和mRNA水平与其来源的胶质瘤恶性度成正相关。3、Nogo-B在GDMECs和胶质瘤肿瘤组织中存在双向表达。4、Nogo-B在胶质瘤中低表达,其促进肿瘤细胞凋亡能力下降,参与了胶质瘤的恶性进展。同时在GDMECs中,高表达的Nogo-B参与了胶质瘤血管的恶性进展。5、由于恶性胶质瘤可以高表达VEGF,VEGF可能通过Nogo-B途径作用于内皮细胞,促进了胶质瘤血管的发生发展,这是Nogo-B在胶质瘤组织和GDMECs中存在双向表达的原因之一。6、Nogo-B siRNA可以抑制内皮细胞增殖,以Nogo-B为靶基因,采用Nogo-B siRNA实行抗胶质瘤血管生成疗法具有一定的理论基础和可行性。
Gliomas are relatively common in the world, these tumors are the leading cause of death from solid tumors in children and the third leading cause of death from cancer in adolescents and adults, most patients with gliomas benefit from microneurosurgery, chemotherapy and radiotherapy. However, despite best therapy and state-of-the-art technology, the mortality rate from gliomas remains high.
Nogo isoforms are part of a superfamily of proteins called reticulons. Nogo-A is a myelin-associated inhibitor of axonal sprouting, Nogo-B is expressed in many cells in culture and is the primary Nogo isoform expressed in blood vessels, and Nogo-C is expressed in the CNS and skeletal muscle. Nogo-A and -B isoforms have a common amino terminus for the first 184 aa, and all three isoforms contain a conserved reticulon homology domain (RHD). A 66-aa loop domain termed Nogo-66 in the RHD can interact with a glycosylphosphatidylinositol-linked cell-surface Nogo-66 receptor (NgR).This receptor mediates, in part, the inhibitory function of Nogo-A on neuronal outgrowth. In contrast to Nogo-A, the amino terminus of Nogo-B (AmNogo-B) promotes the adhesion and chemotaxis of endothelial cells and negatively regulates platelet-derived growth factor-induced chemotaxis insmooth muscle cells. The receptor(s) mediating the actions of Nogo-B are unknown but are necessary to elucidate the potential functions of this ligand. And it was also reported that Nogo-B act as a proapoptotic protein, the loss of which would be typical for cancer cells. Others' research do not support a function of Nogo-B as a physiological pro-apoptotic protein in certain types of cancer.
In this study, we chose to investigate changes in Nogo-B expression in gliomas and glioma derived microvascular endothelial cells (GDMECs), obtained from patients during the microneurosurgical approach, to determine the contribution of Nogo-B in the pathological process of gliomas.
The progress of this study were presented in four parts: 1. In the present study, gliomas and adjacent normal brain tissue without tumorous changes were obtained from patients undergoing microneurosurgery. The mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction, western blotting and immunohistochemistry. 2. GDMECs and normal brain tissue-derived microvascular endothelial cells (MECs) without tumorous changes were obtained from patients undergoing microneurosurgery purified using a MACS magnetic cell sorting system with anti-CD105 antibody. The mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction, western blotting and immunohistochemistry. 3. GDMECs were transfected transiently with VEGF siRNA and the mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction and ELISA. 4. bEnd.3 cells were transfected transiently with Nogo-B siRNA and the mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction and ELISA, then bEnd.3 cells proliferation rate was detected by using the method of WST-1.
The results demonstrated that Nogo-B mRNA expression levels in gliomas decreased compared with levels in normal brain tissues and that protein levels in gliomas decreased also. Tissue Nogo-B immunohistochemical staining in glioma specimens suggested the involvement of Nogo-B in the proliferation of gliomas. The weaker brown staining suggested the lower expression of Nogo-B in gliomas. The results also demonstrated that Nogo-B mRNA expression levels in GDMECs increased compared with levels in MECs and that protein levels in GDMECs. Nogo-B immunohistochemical staining in GDMECs suggested the involvement of Nogo-B in the proliferation of gliomas. Compared with the control GDMECs, VEGF siRNA can inhibit Nogo-B expression significantly. Compared with the control group, Nogo-B siRNA can significantly reduce bEnd.3 cells proliferation in concentration dependent fashion.
These results demonstrated that Nogo-B mRNA and protein expressions are dual-regulated in GDMECs and glioma tissues. It is concluded that Nogo-B protein expression in gliomas and GDMECs is closely correlated to the formation of tumors and that Nogo-B siRNA may inhibit GDMECs proliferation and induce cell apoptosis in order to inhibit tumor growth of gliomas as well.
胶质瘤新生血管的形成在肿瘤发生发展过程中起着重要作用,低度恶性胶质瘤低到中度血管化,而高度恶性的胶质瘤具有较高的血管密度。我们知道,肿瘤扩张必须通过血管生成,即“血管化转换”(Angiogenic switch,AG),以获取氧气和营养,AG是通过肿瘤表达促血管生成因子(包括多肽和蛋白)而完成的,促血管生成因子的生成与环境信号和遗传因子有关,主要包括缺氧、低PH值、低血糖、肿瘤内压力增加、免疫系统所诱发的免疫反应、基因突变等等。
研究表明,胶质瘤血管在结构和功能上都与正常血管不同。例如,肿瘤血管缺乏保护性的生长调节机制,并缺乏有功能的组织特异性血管周细胞。此外,血管并不总是由同源性的内皮细胞层所构成,有时肿瘤细胞和内皮细胞混杂在一起,肿瘤血管管腔差别较大,通透性较高。胶质瘤表现为高度血管化,并且微血管与正常血脑屏障不同,血管通透性增高,由此可引起脑水肿。
此外,胶质瘤脑血管超微结构的研究发现微血管内皮之间紧密连接出现异常,紧密连接蛋白的缺失与血脑屏障的开启密切相关。另外,肿瘤血管形成需要不同的分子信号。例如内皮细胞的趋化和增生信号,细胞基质受体的表达,与细胞外基质降解相关的蛋白酶的表达等。
Nogo-B蛋白是一类中枢髓鞘源性抑制蛋白,是抑制中枢神经元轴突再生的抑制因子,分布广泛,在神经系统中高表达。在外周组织如血管组织、肾脏、肺脏、软骨、肾脏、皮肤、脾中也有表达。
组织损伤后Nogo-B的作用首先表现在参与中枢神经系统轴突再生抑制。Neuron杂志2003年发表了3篇有关Nogo基因缺陷小鼠文章。Strittmatter和同事发现Nogo-A/Nogo-B缺陷小鼠的轴突再生能力改善;Tessier-Lavigne团队分别制造出Nogo-A,B,C3亚型同时缺失模型和Nogo-A,B缺失模型,未发现轴突再生改善;Simonen等人报道伴随Nogo-A缺陷小鼠轴突生长抑制作用降低,中枢神经系统中Nogo-B表达上调。虽然以上文献报道有关Nogo-B功能略有矛盾,但是在某种程度上证明了Nogo-B参与中枢系统损伤后再生。
2004年Nature Medicine上的一篇文章充分揭示了Nogo-B限制外周血管损伤后的再生。Acavedo等人采用质谱分析和RT-PCR等技术首先发现血管的内皮细胞和平滑肌细胞中Nogo-B高表达,进而将其功能片段分别引入这两个细胞系,发现Nogo-B促进内皮细胞的迁移和抑制平滑肌细胞的再生,采用Nogo-B缺失小鼠研究发现,股动脉损伤后平滑肌细胞再生活跃造成血管闭塞,转染Nogo-B后平滑肌增生减弱。
国内Jing-Wei Pan等在2007年发表了Nogo-B和主动脉内皮细胞的关系的文章。文章报道在粥样硬化斑块组织中Nogo-B的mRNA和蛋白表达下调和动脉瘤的形成密切相关,Nogo-B在其中起保护血管的作用。在国外有研究建立了体外细胞模型,发现了Nogo-B更值得关注的功能,其可溶性N端片段对于内皮细胞具有显著的类似VEGF样趋化修复作用,但对于血管平滑肌细胞有阻断PDGF诱导的增生作用。该发现产生了巨大影响,Raines EW在Nature Medicine上作出“Nogo-B为血管病变安装了闸门”的评论,高度评价了Nogo-B在血管病变中的重要功能。
但是Nogo-B在血管病变损伤中的作用机制仍未能完全明确。英国Rousseau S等的意外发现表明,Nogo-B为SAPK2a/p38a通路中MAPKAP-K2的磷酸化底物,提示Nogo-B活性与MAPKs通路密切相关。MAPKs信号通路由一组以级联方式依次活化的丝/苏氨酸蛋白激酶组成,将与细胞膜受体结合的胞外刺激逐级放大并传导到细胞内,调节效应分子参与细胞生长、分化和凋亡等阶段的病理生理活动。其中P38和P42/44已被证明在内皮细胞的迁移和平滑肌细胞增殖中起重要作用。Nogo-B在MAPKs通路中的功能为Nogo-B的机制研究提供了线索,提示Nogo-B对血管的保护作用与MAPKs通路有关。
除了对调节血管生成的作用外,Nogo-B对肿瘤起了到底什么作用?有文献报道Nogo基因表达导致肿瘤细胞凋亡,这个基因在恶性肿瘤细胞系中有一个很强的促凋亡倾向,例如SaOS-2,HT-1080等。这些发现表明Nogo基因是一种新型促凋亡基因。研究表明这些凋亡蛋白位于内质网上,它编码内质网reticulum靶蛋白,其通路和目前已知的前凋亡蛋白不同,在肿瘤细胞中该基因的丢失非常明显。尽管研究表明Nogo-B基因导致凋亡,它不和目前已知的任何一种凋亡相关基因或者功能域同源。相反,它有独特的功能基序,即在它的C末端有一个重复赖氨酸内质保守基序,这个抗凋亡作用与该C-末端分子区域有关,但目前尚不清楚这一作用是否与NgR有关,Nogo-B可与内质网上促凋亡基因Bcl-x1、Bcl-2作用,诱导内质网张力改变和Ca~(2+)内流,影响肿瘤生长。
对于中枢神经系统肿瘤,特别是胶质瘤,目前只有Nogo-A方面的研究内容。Nogo-A的胞外功能域Nogo-66和髓鞘相关糖蛋白(Myelin-associated glycoprotein,MAG)可以让人胶质瘤细胞系U87MG的黏附性和迁移能力显著下降。他认为Nogo-66和MAG可以通过NgR激活,调节胶质瘤生长和迁移,这个现象具有潜在的临床治疗应用价值。但是目前尚没有Nogo-B和胶质瘤的报道。更多的是VEGF、PDGF等常见血管增殖相关生长因子方面的研究。
我们知道VEGF是一种与血管增殖相关生长因子,又称血管通透性因子。其主要生物学活性有:1、促进内皮细胞的增殖。2、增加血管的通透性、维持毛细血管周围内皮细胞的通透性。3、改变细胞外基质,促进血管生长,促进血管构建。4、促进血管形成前暂时性基质沉积。5、选择性激活小动脉、小静脉的内皮细胞,促进内皮细胞分裂、增殖。6、促进内皮细胞的趋化。7、保护新生血管。
已有文献报道恶性肿瘤中肿瘤细胞和血管内皮细胞中VEGF都有表达,且随着肿瘤恶性度的增加,VEGF的表达增加。VEGF主要分布于高度恶性的胶质瘤细胞中和血管内皮细胞,定位于细胞浆,低恶性胶质瘤表达相对较低,正常脑组织几乎检测不出。最新的研究表明肿瘤细胞表面也有VEGF受体高表达,并且受VEGF信号通路的调节,且很多肿瘤高表达VEGF,肿瘤细胞的生长和存活在部分程度上也受VEGF通路的调节。因此阻断VEGF的信号传导通路可以从多个方面抑制肿瘤的生长。目前已有报道采用VEGF单克隆抗体、VEGF受体拮抗剂等抑制肿瘤生长,最近还有利用RNA干扰技术,利用VEGF siRNA实行抗肿瘤血管生成疗法的报道,取得了一定进展。
RNA干扰(RNA interference,RNAi)是指细胞中导入与内源性mRNA编码区某段序列同源的双链RNA(dsRNA),可致该mRNA发生特异性降解从而导致基因表达沉默的现象。因基因沉默现象发生在转录后水平,又称为转录后基因沉默(PTGS)。目前,RNAi技术作为一个强有力的遗传学工具,提供了高效、特异性的抑制基因表达的手段,成为基因功能研究、抗病毒感染研究和基因治疗研究的重要方法。
恶性胶质瘤的发生、发展离不开微血管内皮细胞,微血管内皮细胞同样受到恶性胶质瘤细胞分泌的各种细胞因子的作用。目前对这些因子中的Nogo-B蛋白的研究主要集中于周围血管的内皮细胞上,在中枢神经系统中,Nogo-B对胶质瘤细胞和胶质瘤微血管内皮细胞到底起了什么样的作用,Nogo-B和VEGF之间到底存在什么样的关系,VEGF是否参与了Nogo-B的调节通路,如何利用Nogo-B和VEGF实行抗肿瘤血管生成疗法,这些是本课题的研究内容和目的。
研究分四个部分,第一部分:人胶质瘤组织中Nogo-B的功能表达。第二部分:人胶质瘤源性微血管内皮细胞中Nogo-B的功能表达。第三部分:VEGF参与胶质瘤微血管内皮细胞Nogo-B表达的调控机制。第四部分:干扰Nogo-B表达抑制微血管内皮增生。
研究采用以下研究方法:1、免疫组织化学、Western blotting法、Real-time PCR法分析在不同级别的胶质瘤组织中Nogo-B在蛋白和mRNA水平上的功能表达。2、利用免疫磁珠内皮细胞分选系统纯化胶质瘤源性微血管内皮细胞(Glioma derivedmicrovascular endothelial cells,GDMECs)。3、利用免疫组织化学法、Western blotting法、Real-time PCR法分析Nogo-B在不同级别的GDMECs中的功能表达。4、将VEGFsiRNA瞬时转染至GDMECs中,采用ELISA法、Real-time PCR法分析VEGF对GDMECs中Nogo-B表达的功能调控。5、采用小鼠脑血管内皮细胞系bEnd.3,将Nogo-B siRNA和bEnd.3共培养,观察bEnd.3增殖情况,探讨以Nogo-B作为靶点实行抗胶质瘤血管生成疗法的可行性。
本研究得到如下结果:1、人胶质瘤组织中Nogo-B与正常脑组织相比,染色阳性率降低。2、在高级别的胶质瘤中Nogo-B在蛋白和mRNA水平上表达下调,Nogo-B表达水平随着肿瘤恶性程度增加而显著降低。3、在GDMECs中,Nogo-B蛋白、mRNA表达强度随着其来源胶质瘤的恶性程度增加而显著增加。4、瞬时转染VEGFsiRNA到GDMECs后,Real-time PCR、ELISA结果表明干扰VEGF表达能显著促进降低Nogo-B的蛋白表达。5、Nogo-B siRNA和bEnd.3细胞共孵育后,bEnd.3中Nogo-B表达显著抑制,其抑制程度具有siRNA剂量依赖性;抑制Nogo-B表达后,和对照组相比,bEnd.3增殖受到抑制。
本研究得出结论:1、在人胶质瘤组织中,Nogo-B在蛋白和mRNA水平的表达与肿瘤恶性度成负相关。2、在GDMECs中,Nogo-B在蛋白和mRNA水平与其来源的胶质瘤恶性度成正相关。3、Nogo-B在GDMECs和胶质瘤肿瘤组织中存在双向表达。4、Nogo-B在胶质瘤中低表达,其促进肿瘤细胞凋亡能力下降,参与了胶质瘤的恶性进展。同时在GDMECs中,高表达的Nogo-B参与了胶质瘤血管的恶性进展。5、由于恶性胶质瘤可以高表达VEGF,VEGF可能通过Nogo-B途径作用于内皮细胞,促进了胶质瘤血管的发生发展,这是Nogo-B在胶质瘤组织和GDMECs中存在双向表达的原因之一。6、Nogo-B siRNA可以抑制内皮细胞增殖,以Nogo-B为靶基因,采用Nogo-B siRNA实行抗胶质瘤血管生成疗法具有一定的理论基础和可行性。
Gliomas are relatively common in the world, these tumors are the leading cause of death from solid tumors in children and the third leading cause of death from cancer in adolescents and adults, most patients with gliomas benefit from microneurosurgery, chemotherapy and radiotherapy. However, despite best therapy and state-of-the-art technology, the mortality rate from gliomas remains high.
Nogo isoforms are part of a superfamily of proteins called reticulons. Nogo-A is a myelin-associated inhibitor of axonal sprouting, Nogo-B is expressed in many cells in culture and is the primary Nogo isoform expressed in blood vessels, and Nogo-C is expressed in the CNS and skeletal muscle. Nogo-A and -B isoforms have a common amino terminus for the first 184 aa, and all three isoforms contain a conserved reticulon homology domain (RHD). A 66-aa loop domain termed Nogo-66 in the RHD can interact with a glycosylphosphatidylinositol-linked cell-surface Nogo-66 receptor (NgR).This receptor mediates, in part, the inhibitory function of Nogo-A on neuronal outgrowth. In contrast to Nogo-A, the amino terminus of Nogo-B (AmNogo-B) promotes the adhesion and chemotaxis of endothelial cells and negatively regulates platelet-derived growth factor-induced chemotaxis insmooth muscle cells. The receptor(s) mediating the actions of Nogo-B are unknown but are necessary to elucidate the potential functions of this ligand. And it was also reported that Nogo-B act as a proapoptotic protein, the loss of which would be typical for cancer cells. Others' research do not support a function of Nogo-B as a physiological pro-apoptotic protein in certain types of cancer.
In this study, we chose to investigate changes in Nogo-B expression in gliomas and glioma derived microvascular endothelial cells (GDMECs), obtained from patients during the microneurosurgical approach, to determine the contribution of Nogo-B in the pathological process of gliomas.
The progress of this study were presented in four parts: 1. In the present study, gliomas and adjacent normal brain tissue without tumorous changes were obtained from patients undergoing microneurosurgery. The mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction, western blotting and immunohistochemistry. 2. GDMECs and normal brain tissue-derived microvascular endothelial cells (MECs) without tumorous changes were obtained from patients undergoing microneurosurgery purified using a MACS magnetic cell sorting system with anti-CD105 antibody. The mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction, western blotting and immunohistochemistry. 3. GDMECs were transfected transiently with VEGF siRNA and the mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction and ELISA. 4. bEnd.3 cells were transfected transiently with Nogo-B siRNA and the mRNA and protein expression levels of Nogo-B were measured with real-time polymerase chain reaction and ELISA, then bEnd.3 cells proliferation rate was detected by using the method of WST-1.
The results demonstrated that Nogo-B mRNA expression levels in gliomas decreased compared with levels in normal brain tissues and that protein levels in gliomas decreased also. Tissue Nogo-B immunohistochemical staining in glioma specimens suggested the involvement of Nogo-B in the proliferation of gliomas. The weaker brown staining suggested the lower expression of Nogo-B in gliomas. The results also demonstrated that Nogo-B mRNA expression levels in GDMECs increased compared with levels in MECs and that protein levels in GDMECs. Nogo-B immunohistochemical staining in GDMECs suggested the involvement of Nogo-B in the proliferation of gliomas. Compared with the control GDMECs, VEGF siRNA can inhibit Nogo-B expression significantly. Compared with the control group, Nogo-B siRNA can significantly reduce bEnd.3 cells proliferation in concentration dependent fashion.
These results demonstrated that Nogo-B mRNA and protein expressions are dual-regulated in GDMECs and glioma tissues. It is concluded that Nogo-B protein expression in gliomas and GDMECs is closely correlated to the formation of tumors and that Nogo-B siRNA may inhibit GDMECs proliferation and induce cell apoptosis in order to inhibit tumor growth of gliomas as well.
引文
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