【作者】 廖艺玮；

【导师】 袁贤瑞；

【作者基本信息】 中南大学 ， 外科学， 2009， 博士

【摘要】 背景:胶质瘤是中枢神经系统内最常见的恶性肿瘤,占颅内肿瘤的40%-50%。其呈侵袭性生长,总体疗效不佳,尤其是高分级胶质瘤,具有高度问变的生长特点,术后复发快、预后差,严重威胁人类健康,是神经外科治疗中相对棘手的难治性肿瘤之一。增殖的微血管对胶质瘤的恶性生物学行为起着重要作用。在胶质瘤的分级标准中,将血管的形态学改变作为恶性指标之一,明显不同于颅外其他肿瘤,无疑将血管生成在该瘤中的作用上升到一个新的高度。目前抗血管生成作为肿瘤治疗的新方法,已迅速发展为重要的抗癌策略,并成为肿瘤治疗领域的研究热点。在经手术切除、放疗和化疗等综合治疗仍无法从根本上改善胶质瘤预后的情况下,抗血管生成治疗有可能成为改善胶质瘤预后的突破口。fibulin-5是细胞外基质蛋白家族fibulin家族中的成员,亦称为FBLN-5、DANCE或EVEC,在人染色体中位于14q32.1,其蛋白大小约60KDa。fibulin-5是一种含有448个氨基酸的糖蛋白,含有6个与钙离子结合的表皮生长因子样（EGF-like）区域,其中一个区域含有能与整合素结合的RGD基序（integrin-binding RGD motif）。后者能介导内皮细胞粘附,能与细胞表面的整合素αvβ3、αvβ5和α9β1结合。fibulin-5广泛分布于富含弹性蛋白的组织中,能直接与弹性蛋白原（tropoelastin）结合,或者连接微原纤维（microfibril）,在形成弹性纤维过程中具有重要作用,而且可以影响血管的发育和修复。此外,fibulin-5还能通过促进组织肉芽增生和Ⅰ型胶原表达来促进伤口愈合;这与细胞的增殖、运动和侵袭有关。因此既往对其研究主要集中在弹性蛋白病变方面,对其缺失或基因突变而导致的多种皮肤、血管和肺组织疾病已经有了充分的共识,认为它是治疗血管硬化、肺气肿和皮肤松弛症等疾病的重要靶点。有研究指出fibulin-5可以促进人HT1080纤维肉瘤细胞的DNA合成,并增强其侵袭基底膜的能力;但是在肾癌、乳腺癌、卵巢癌和结肠癌等恶性肿瘤中,fibulin-5的表达明显降低。另有实验发现fibulin-5能通过抑制肿瘤血管生成来减缓老鼠癌性肿瘤的生长。这些研究显示fibulin-5的表达与肿瘤发生、发展和血管生成存在紧密联系。但是目前fibulin-5对肿瘤细胞的生物学特性的影响还不甚清楚;在肿瘤血管生成中的具体调控机制还有待进一步研究;在胶质瘤中的表达及作用在国内外亦未见到相关报道。HUVEC-2C是经典的血管内皮细胞模型,U251是来源于多形性胶质母细胞瘤WHOⅣ级的细胞株。因此在本文中,我们拟首先探讨fibulin-5在人脑胶质瘤中的表达情况。然后拟通过体外单纯血管内皮细胞模型（HUVEC-2C）以及Transwell培养技术构建的HUVEC-2C和U251共培养的胶质瘤微环境模型探讨fibulin-5与胶质瘤发生、发展的关系。第一章fibulin-5在人脑胶质瘤组织中的表达及意义目的:初步探讨fibulin-5在胶质瘤中的表达情况及其与胶质瘤发生、发展的关系。方法:通过免疫组织化学方法检测fibulin-5蛋白、Ki-67,FactorⅧ及HIF-1α蛋白在正常脑组织和胶质瘤组织中的表达情况,并分析fibulin-5蛋白的表达与胶质瘤的病理分级、胶质瘤Ki-67标记指数（Ki-67 LI）、微血管密度（MVD）及HIF-1α蛋白表达的关系。实验分三组:对照组为正常脑组织（7例）、低分级胶质瘤组（21例WHO分类Ⅰ～Ⅲ级的胶质瘤石蜡标本）和高分级胶质瘤组（19例WHO分类Ⅲ～Ⅳ级的胶质瘤石蜡标本）。通过RT-PCR检测fibulin-5 mRNA在正常脑组织和胶质瘤组织中的表达情况。实验分三组:对照组为正常脑组织（7例）、低分级胶质瘤组（22例WHO分类Ⅰ～Ⅱ级的新鲜胶质瘤标本）和高分级胶质瘤组（15例WHO分类Ⅲ～Ⅳ级的新鲜胶质瘤标本）。结果:fibulin-5蛋白主要分布于细胞之间,偶可在实质细胞及血管内皮细胞胞浆中发现表达。均表现为黄色或棕黄色着色。fibulin-5蛋白在对照组正常脑组织中,所有7例标本均检测到表达;在低分级胶质瘤组中,21例标本有13例检测到表达;在高分级胶质瘤组中,19例标本有4例检测到表达。正常脑组织标本的fibulin-5蛋白表达阳性率以及平均IRS值和低分级胶质瘤组相比无统计学差异（P＞0.05）;高分级胶质瘤组的fibulin-5蛋白表达阳性率以及平均IRS值均低于正常脑组织和低分级胶质瘤组的表达,统计学检测有差异（P＜0.05）。在有经验的病理科医师指导下,观察实验各组的阳性标本,可以发现fibulin-5蛋白在组织血管中表达的位置是不一样的,在正常脑组织中主要表达于管径较大的血管;在低分级胶质瘤中主要表达于管径微小的血管;在高分级胶质瘤中未发现在组织血管表达。fibulin-5 mRNA在对照组正常脑组织中,所有7例标本均检测到表达;在低分级胶质瘤组中,22例标本有14例检测到表达;在高分级胶质瘤组中,15例标本有3例检测到表达。正常脑组织标本的fibulin-5 mRNA表达阳性率以及平均IOD值和低分级胶质瘤组相比无统计学差异（P＞0.05）;高分级胶质瘤组的fibulin-5 mRNA表达阳性率以及平均IOD值均低于正常脑组织和低分级胶质瘤组的表达,统计学检测有差异（P＜0.05）。在胶质瘤组织中,fibulin-5蛋白和mRNA在不同性别及年龄的表达无统计学差异（P＞0.05）。同时直线相关分析发现,fibulin-5蛋白在胶质瘤中的表达与胶质瘤的病理分级、MVD、HIF-1α及Ki-67均存在负相关（r=-0.455,-0.779,-0.320;P=0.003,0.000,0.044）。结论:fibulin-5在正常脑组织中表达,其在胶质瘤组织中表达的变化可能与胶质瘤的进展和血管生成存在联系。第二章构建人fibulin-5基因表达的慢病毒质粒并感染HUVEC—2C筛选混合稳定株目的:构建人fibulin-5基因表达的慢病毒质粒并感染HUVEC—2C筛选混合稳定株,为体外研究fibulin-5的生物学功能提供材料。方法:通过RT-PCR检测fibulin-5 mRNA在HUVEC-2C和U251细胞株的表达。通过构建含有fibulin-5的重组pGC-FU载体,并用293T细胞包装病毒,将得到的人fibulin-5基因表达的慢病毒质粒感染HUVEC-2C并筛选,最终得到人fibulin-5基因表达的HUVEC-2C混合稳定株。结果:fibulin-5 mRNA在HUVEC-2C和U251细胞株均不表达。在人fibulin-5基因表达的HUVEC-2C混合稳定株中,通过Western blot和Real-time PCR方法检测到fibulin-5的表达。结论:成功建立了人fibulin-5基因表达的HUVEC-2C混合稳定株,为进一步实验做准备。第三章人fibulin-5基因表达对内皮细胞生物学特性的影响目的:探讨人fibulin-5基因表达对内皮细胞生物学特性的影响,以明确其在血管生成中的作用及机制。方法:实验分3组:CON组（未作任何干扰的HUVEC-2C细胞株）;NC组（阴性对照慢病毒感染HUVEC-2C筛选的混合稳定细胞株）;KD组（人fibulin-5基因表达的慢病毒质粒感染HUVEC-2C筛选的混合稳定细胞株）。通过MTT法、结晶紫染色法、流式细胞仪分析法和成管能力实验检测人fibulin-5基因表达对HUVEC-2C的增殖、粘附、迁移、凋亡和成管能力的影响。结果:MTT检测发现在相同的时问点,KD组的OD值明显低于CON组和NC组（P＜0.05）;检测粘附能力试验中,接种30min后KD组结晶紫OD值明显高于CON组和NC组（P＜0.05）;检测迁移能力实验中,KD组结晶紫OD值明显低于CON组和NC组（P＜0.05）;培养至第3天进行流式细胞仪检测可见,KD组HUVEC细胞G0/G1期分布明显多于NC组和CON组（P＜0.05）;S期分布明显少于NC组和CON组（P＜0.05）:G₂/M期分布和细胞凋亡率三组无明显差别（P＞0.05）。检测成管能力实验中,CON组、NC组及KD组管状结构相对面积并无明显差异（P=0.305）,但是KD组HUVEC-2C成管数明显低于CON组和NC组（P<0.05）。在检测增殖、粘附、迁移、凋亡及成管能力实验中,CON组和NC组无明显差异（P>0.05）。结论:人fibulin-5基因表达后,可以使内皮细胞的细胞周期迟滞;抑制内皮细胞的增殖、迁移和成管能力;可以增强内皮细胞的粘附能力。其可能通过减少血管基底膜的溶解、维持细胞外基质的稳定性、增强内皮细胞的粘附能力来抑制血管新生和维护原有血管的稳定。第四章人fibulin-5基因表达对胶质瘤-内皮细胞微生态系统中内皮细胞和肿瘤细胞生物学特性的影响目的:探讨人fibulin-5基因在胶质瘤微环境中表达对内皮细胞和肿瘤细胞生物学特性的影响,以明确其在胶质瘤发生、发展中的作用。方法:用Transwell技术体外建立了U251细胞与HUVEC共培养模型。实验分3组:CON组（其中HUVEC-2C未加任何干扰因素）;NC组（其中HUVEC-2C是阴性对照慢病毒感染HUVEC-2C筛选的混合稳定细胞株）;KD组（其中HUVEC-2C是人fibulin-5基因表达的慢病毒质粒感染HUVEC-2C筛选的混合稳定细胞株）。通过MTT法、结晶紫染色法和成管能力实验检测人fibulin-5基因在胶质瘤微环境中表达对HUVEC-2C的增殖、粘附、迁移和成管能力的影响。通过MTT法和结晶紫染色法检测人fibulin-5基因在胶质瘤微环境中表达对U251的增殖和迁移能力的影响。结果:①在检测人fibulin-5基因在胶质瘤微环境中表达对HUVEC-2C生物学功能影响的实验中发现:MTT法结果示在相同的时间点,KD组的OD值明显低于CON组和NC组（P＜0.05）;检测粘附能力试验中,接种30min后KD组结晶紫OD值明显高于CON组和NC组（P＜0.05）;检测迁移能力实验中,KD组结晶紫OD值明显低于CON组和NC组（P＜0.01）;检测成管能力实验中,CON组,NC组及KD组管状结构相对面积并无明显差异（P=0.996）,但是KD组HUVEC-2C成管数明显低于CON组和NC组（P＜0.05）,KD组的管型结构更加规则。在检测增殖、粘附、迁移及成管能力实验中,CON组和NC组无明显差异（P＞0.05）。②在检测人fibulin-5基因在胶质瘤微环境中表达对U251生物学功能影响的实验中发现:MTT法结果示从培养48小时后开始,在相同的时间点,KD组的OD值明显低于CON组和NC组（P＜0.05）,CON组和NC组的OD值无明显差异（P＞0.05）;检测迁移能力实验中,CON组、NC组和KD组结晶紫OD值比较无明显差异（P=0.819）结论:人fibulin-5基因在胶质瘤微环境中表达可抑制内皮细胞的增殖、迁移和成管能力;可以增强内皮细胞的粘附能力。其可抑制血管新生和维护原有血管的稳定,并有可能使“肿瘤血管相对正常化”;此外,它还可以抑制U251的增殖,从而抑制肿瘤的增殖和转移。更多还原

【Abstract】 BACKGROUND:Gliomas,especially the high-grade gliomas,are the most common primary brain malignant tumor whose characteristic is quickly recurrence and an extremely poor prognosis,despite advances in surgical and clinical neuro-oncology.The microvessel density is an independent index in judging the grade of gliomas as to the conclusive evidence.Angiogenesis maybe the most important factor for the proliferation and metastasis of gliomas,and the anti-angiogenesis treatment maybe the most worth expectation for the better prognosis of the patients who had got the gliomas.fibulin-5（FBLN-5,DANCE or EVEC）,which is a 448 amino acid glycoprotein,is the member of the fibulin family of extracellular matrix （ECM） proteins,and is mapped in chromosome 14q32.1.It contains six EGF-like repeats,all containing consensus sequences for Ca²⁺-binding（CB）-EGF-like repeats.The first CB-EGF-like repeat of fibulin-5 also contains a RGD sequence,which mediates interaction with cell surface integrins,includingαvβ3、αvβ5 andα9β1.fibulin-5 is a multifunctional extracellular matrix protein that mediates cell-cell and cell-matrix communication.It regulates the formation and assembly of elastic fibers primarily by binding tropoelastin, and secondarily by binding lysyl oxidase-like 1（Loxl-1） and EMILIN-1 （elastin microfibril interface-located protein）.It can enhanced wound closure and healing by increasing tissue granulation and type I collagen expression.Its expression is normally low in adult vasculature,but it is remarkably elevated in developing or injured blood vessels,and in uterine myometrial arteries undergoing cyclic angiogenesis.Also,it takes an important role in the angiosclerosis and cutis laxa.The expression of fibulin-5 also augments the tumorigenicity of human HT1080 fibrosarcoma cells by increasing their DNA synthesis, migration toward fibronectin,and invasion through synthetic basement membranes.In stark contrast,Its expression is down-regulated in the majority of metastatic human malignancies,particularly in cancers of the kidney,breast,ovary,and colon.It also can slow the growth of cancer tumors in mice by preventing blood vessels from sprouting,fibulin-5 may play an important part in the tumor procession.In our research,we will discuss the role that fibulin-5 plays in the process of gliomas.Part 1 Expression and function of fibulin-5 gene in the gliomasObjective:The present study was carried out to investigate the expression of fibulin-5 in human glioma tissues.Methods:40 cases of formalin-fixed,paraffin-embedded specimens of tissue,including low-grade glioma of 21 patients（gradeⅠ～Ⅱ） and high-grade glioma of 19 patients（gradeⅢ～Ⅳ）,and another normal brain tissues of 7 patients, were used to assess the expression status of fibulin-5 protein,Ki-67, FactorⅧand HIF-1αby immunohistochemical staining.The Ki-67 labeling index（LI） and microvessel density（MVD） were calculated. Then we also analyzed the correlation between fibulin-5’s expression and the Ki-67 LI,MVD or the expression of HIF-1α.Another 44 cases of the fresh tissue,including low-grade glioma of 22 patients（gradeⅠ～Ⅱ）, high-grade glioma of 15 patients（gradeⅢ～Ⅳ） and the normal brain tissues of 7 patients,were used to assess the expression status of fibulin-5 mRNA by RT-PCR.Results:The expression of the fibulin-5’s protein usually lay between cell and cell or cell and matrix.In the normal brain tissue,it always could be found;in the low grade gliomas tissue,there were 13 sample could be found;in the high grade gliomas,there were only 4 sample could be found.The expression of the fibulin-5’s protein in the normal brain tissues and the low grade gliomas were higher than its expression in the high grade gliomas（P＞0.05）.The expression of fibulin-5’s protein in the vessel which was in the normal brain tissue usually lay in the great vessel,but in the low grade tissue,it usually lay in the capillary vessel.The expression of the fibulin-5’s protein inverse correlated with the degree of malignancy,Ki-67 LI,MVD and the expression of HIF-1αof human gliomas（r=-0.455,-0.779,-0.320;P =0.003,0.000,0.044 ）.Then we found that fibulin-5’s mRNA always expressed in the normal brain tissue;there were 14 sample could be found in the low grade gliomas tissue;there were only 3 sample could be found in the high grade gliomas tissue.The expression of the fibulin-5’s mRNA in the normal brain tissues and the low grade gliomas were higher than its expression in the high grade gliomas（P＞0.05）.Conclusion:fibulin-5 could expressed in the normal brain tissue.The expression of fibulin-5 may correlated with the process and the angiogenesis of gliomas.Part 2 To construct the cell strain（HUVEC-2C） for expressing the fibulin-5 gene through the stable transfectionObjective:To construct an lentiviral vector carrying fibulin-5 gene and then stable transfected to the huaman umbilical vein endothelial cells -2C（HUVEC-2C）.Methods:The expression of fibulin-5 in the HUVEC-2C and the U251 was checked by RT-PCR first.Then fibulin-5 was amplified by PCR and subcloned fibulin-5 into the lentiviral vector（pGC-FU）,to generate the lentiviral expression vector, （pGC-FU-FIBULIN-5）.Recombinant lentiviruses were produced by 293T cells following the co-transfection of pGC-FU-FIBULIN-5,with the packaging plasmids pHelper1.0 and pHelp2.0.So,we got the lentiveruses called KL425-LV.Last,we use the KL425-LV to stable transfect into HUVEC-2C and got the cell strain called Fibulin-5-GFP-LV. Results:There was no expression in the HUVEC-2C and the U251.The cell strain（Fibulin-5-GFP-LV） was expressing the fibulin-5 gene through the check by Western blot and Real-time PCR.Conclusion:The cell strain which was expressing the fibulin-5 gene was successed constructing.Part 3 Effect on the bionomics of HUVEC-2C by the fibulin-5 gene expressingObjective:The present study was carried out to investigate the effect on the bionomics of HUVEC-2C by the fibulin-5 gene expressing. Then we could figure out the function of the fibulin-5 during the angiogenesis.Methods:There were three groups in the experiment:CON group（used the normal HUVEC-2C）,NC group（used the HUVEC-2C which was transfected the negative vector） and KD group（used the cell strain called Fibulin-5-GFP-LV,which was contructed in the Part 2）.So we could exam the change of the ability of the proliferation,adhesion, invasion,apoptosis and vessel-sprouting of the HUVEC-2C during the fibulin-5 gene expressing.Results:By the MTT test,we found that the OD value in the KD group was lower than that in the CON group and the NC group（P＜0.05）.In the adhesion’s test,the OD value in the KD group was higher than that in the CON group and the NC group（P＜0.05）.In the invasion’s test,we found that the OD value in the KD group was lower than that in the CON group and the NC group（P＜0.05）.By the flow cytometry’s test,we found that the cell cycle in the KD group was prolonged（P＜0.05）.In the vessel-sprouting abiltity test,we found that the number of the vessel was decreased in the KD group（P＜0.05）. Conclusion:The expression of fibulin-5 gene could prolong the period of the cell cycle,inhibit the ability of the proliferation,invasion and the vessel-sprouting of HUVEC-2C.But it could enhance the ability of the adhesion,fibulin-5 gene expressing could inhibit the angiogenesis and make the vessel more stable.Part 4 Effect on the bionomics of HUVEC-2C and U251 by fibulin-5 gene expressing in the gliomas’s microenvironmentObjective:The present study was carried out to investigate the effect on the bionomics of HUVEC-2C or U251 by the fibulin-5 gene expressing in the gliomas’s microenvironment.Then we could figure out the function of the fibulin-5 during the process of gliomas.Methods:We first constructed the gliomas’s microenvironment by HUVEC-2C and U251 co-cultured.There were three groups in the experiment:CON group（used the normal HUVEC-2C）,NC group（used the HUVEC-2C which was transfected the negative vector） and KD group（used the cell strain called Fibulin-5-GFP-LV,which was contructed in the Part 2）.So we could exam the change of the ability of the proliferation,adhesion, invasion and vessel-sprouting of the HUVEC-2C during the fibulin-5 gene expressing in the gliomas’s microenvironment.We also examed the change of ability of the proliferation and invasion of the U251 by the same way.Results:①Testing the change of the ability of HUVEC-2C’s bionomics in the gliomas’s microenvironment:By the MTT test,we found that the OD value in the KD group was lower than that in the CON group and the NC group（P＜0.05）.In the adhesion’s test,the OD value in the KD group was higher than that in the CON group and the NC group （P＜0.05）.In the invasion’s test,we found that the OD value in the KD group was lower than that in the CON group and the NC group（P＜0.05）. In the vessel-sprouting ability test,we found that the number of the vessel was decreased in the KD group（P＜0.05）.②Testing the change of the ability of U251’s bionomics in the gliomas’s microenvironment:By the MTT test,we found that the OD value in the KD group was lower than that in the CON group and the NC group（P＜0.05）.In the invasion’s test, we found that there were no difference in the KD group,CON group and NC group（P＞0.05）.Conclusion:The expression of fibulin-5 gene in the gliomas’s microenvironment could inhibit the ability of the proliferation, invasion and the vessel-sprouting of HUVEC-2C.It could enhanced the ability of the adhesion of HUVEC-2C.It could also inhibit the U251 ’s reproductive activity,fibulin-5 gene expressing could inhibit the angiogenesis and make the vessel more stable in the gliomas’s microenvironment.It mightbe make the tumor vessel normalization. Mightbe,it inhibit the gliomas’s ability of the proliferation and the invasion directly.更多还原