【作者】 李亮；

【导师】 郭宗泽；

【作者基本信息】 中国医科大学 ， 外科学， 2008， 硕士

【摘要】 目的胶质瘤是颅内最常见的恶性肿瘤,约占成人原发性颅内肿瘤的50—60%。目前以手术治疗为主,由于其呈弥漫侵润性生长,与正常脑组织分界不清楚,难以做到彻底切除,术后虽结合放、化疗,但由于其对放化疗敏感性差,多数患者仍就死于胶质瘤的复发或进展。病理学研究发现恶性胶质瘤常伴缺氧区域,甚至中心坏死以及周围异常血管的增生,为深入研究其分子学发生的机制,本实验研究相关缺氧因子HIF-1α和nNOS在人脑胶质瘤中表达情况,以此探讨HIF-1α和nNOS与胶质瘤异质性之间的联系,以及二者的关联性,从而为胶质瘤在低氧血管生成方面的研究提供理论依据,为下一步治疗胶质瘤奠定理论基础。实验方法方法:(1)研究对象:选择在2007年6月至2007年12月期间,于中国医科大学附属一院神经外科就治,并经手术切除,术后病理证实为胶质瘤(包括胶质瘤Ⅰ、Ⅱ、Ⅲ、Ⅳ级)30例。根据WHO(1999年)关于人脑胶质瘤病理分类方法进行分类分组。(2)免疫组织化学方法鉴定HIF-1α和nNOS在不同病理级别人脑胶质瘤中表达情况:冰冻切片经过氧化物酶阻断剂阻断内源性过氧化物的活性,室温孵育30分钟后,加入动物血清孵育30分钟后,加入一抗4℃过夜,第二日加入二抗室温孵育60分钟,滴加辣根酶标记联酶卵白素工作液孵育60分钟,采用DAB方法染色,最后苏木素复染,中性胶固定封片。(3)图像分析与处理:规定HIF-1α和nNOS染色的半定量评判标准,采用人工观察的方法计数。采用自动图像分析法测定平均光密度值。实验数据采用SPSS13.0 For Windows软件进行统计学处理。实验结果染色结果显示所有胶质瘤均有不同程度HIF-1α和nNOS的表达。HIF-1α主要在细胞核深染,nNOS主要在细胞质深染,二者的表达程度与病理级别有关,高级别胶质瘤组的表达明显高于低级别组的表达,统计学显示其分布差异显著(P＜0.01)。同时应用自动图像分析法测定平均光密度值显示:不同级别胶质瘤HIF-1α和nNOS的表达有明显差异(P＜0.01),并与病理级别呈正相关,HIF-1α和nNOS二者的表达亦有相关性。讨论胶质瘤是颅内最常见的恶性肿瘤,肿瘤细胞的侵袭和侵润是胶质瘤的特征之一,也是临床手术全切困难,易引起复发的基础。病理学研究发现恶性胶质瘤常伴缺氧区域,甚至中心坏死以及周围异常血管的增生。近来研究表明HIF-1α作为肿瘤微环境中的一个至关重要的调节因子,在促进肿瘤血管生成和肿瘤的侵袭性方面起中心作用。HIF-1α过度表达,它与其下游基因(如VEGF中的缺氧反应元件,Hypoxia response elements,HRE)中的共有序列结合,在转录水平上增加VEGF的表达。HIF-1α在低氧环境中的显著增加VEGF mRNA的稳定性,从而上调VEGF蛋白表达水平,增加缺氧区域血管的生成。本研究证实了HIF-1α在胶质瘤细胞中的表达,并且随着胶质瘤病理级别的升高HIF-1α表达增强。另外,近来有报道nNOS在恶性胶质瘤低氧血管生成方面也发挥重要的作用,nNOS通过cGMP途径开放血脑屏障,提高VEGF水平,增加新生血管形成,同时一氧化氮扩张血管,增加血管血液流量,从而满足肿瘤细胞对于氧的需求。本研究证实了nNOS在胶质瘤细胞中的表达,并且随着胶质瘤病理级别的升高nNOS表达增强。证实了HIF-1α和nNOS在恶性胶质瘤中的表达存在相关性,推测二者共同参与脑胶质瘤血管生成,提高胶质瘤增殖和侵袭能力。结论HIF-1α和nNOS的表达与脑胶质瘤病理级别密切相关,HIF-1α和nNOS的表达随病理级别的升高表达增强,二者表达也呈正相关,故二者表达定量评估可做为脑胶质瘤恶性程度分级指标和胶质瘤病人预后判断指标之一。更多还原

【Abstract】 ObjectiveGlioma is one of the most common intracranial malignant tumors and it reaches to the 50-60% of the total intracranial tumors. Surgical excision is a chief treatment of glioma currently; however, it’s hard to excise the glioma thoroughly, since between which and the normal tissues there is no border-line, due to it grows in a diffused and infiltrated way. Although combining with radiotherapy and chemotherapy after surgical treatment, most patients died of progression or recurrence of glioma, for which is insensitive to radio- or chemo-therapy. The pathological studies found that, there are anoxic zones, central necrosis and proliferation of abnormal vessels around the malignant glioma. For the purpose of investigating the molecular mechanism of glioma further, in this study, we examined the expression of hypoxia inducible factor 1 alpha (HIF-1α) and neurological nitric oxide synthase (nNOS) in human glioma; and then we discussed not only the roles of HIF-1 a and nNOS in heterogeneity of glioma but also the correlation between them. Our study would provide a basic theory to researches on the mechanisms of hypoxia angiogenesis in glioma, and establish new ideas for further treatment of the most common intracranial malignant tumor.MethodSubject: 30 cases of excised human glioma tissue samples (including grade I、II、III、IV), proved by post-operational pathological results, were obtained from the patients in charged in the Neurosurgery of the First Affiliated Hospital of China Medical University from June 2007 to December 2007. According to the pathological classification criteria of human glioma published by WHO in 1999, the samples were divided into two groups.Immunohistochemical stain was used to identify the expression of HIF-1 a and nNOS in different pathological grade of human glioma, respectively. Firstly, peroxidase inhibitors were used to inactivate the endogenesic peroxidate in frozen slices for incubating at room temperature for 30 minutes. After that the samples were washed with PBS and incubated with animal-serum for 30 minutes, then added with primary antibody and incubated at 4℃overnight. In the following day, the samples were added with second antibody and incubated for 60 minutes at room temperature. Then the samples were added with Streptavidin-horseradish peroxidase and incubated at room temperature for 60 minutes. Finally, the samples were stained by DAB method, and counterstained with haematoxylin. After that, the samples were fixed with resin.The semi-quantification criterion of the stain of HIF-1αand nNOS was identified, naked-eye observing method was used for cell counting, and automatic graphic analysis system was used to exam the optical density of a specified field. We processed the acquired data using the statistic software SPSS13.0 for Windows.ResultOur results of the staining suggest that, there are Hif-1αand nNOS expression in glioma tissues of all grades. Hif-1αis mainly stained in nuclear and nNOS is mainly stained in cytoplasm, respectively; and the expressions of both of them are correlated to the pathological grades. The expression level of higher grades glioma is obviously higher than the lower grades, and there is an obvious statistical distributional difference (P<0.01). Moreover, the result of average optical density determined by an automatic graphic analysis system shows that, there is an obvious distinction among the expression levels of Hif-1αand nNOS in different grades of glioma, and which are positively correlated with the pathological grades. And also we found that there is a relationship between the expression of Hif-1αand nNOS.DiscussionGlioma is one of the most common intracranial malignant tumors, and one of whose features is the invasion and infiltration of tumor cells, which is also the basic reason for recurrence and residual of tumor after surgical excision. The pathological studies found that, there are anoxic zones, central necrosis and proliferation of abnormal vessels around the malignant glioma. In recent studies, Hif-1αwas identified to be a most important regulator in the micro-circumstance of tumor cell, playing a central role in angiogenesis and invasion of glioma. The over expression of Hif-1αwill increase the expression of VEGF in transcriptional level, because Hif-1αcan combine with the consensus sequence in its downstream genes (i.e. hypoxia response elements in VEGF, HRE). Hif-1αcan increase the stability of VEGF mRNA obviously in hypoxia circumstances, make the expressing level of VEGF protein up-regulated, and therefore proliferate generation of vessels in hypoxia regions. This study demonstrated that Hif-1αis expressed in glioma cells and the expression level of Hif-1αis increased with the pathological grade of glioma accordingly. Furthermore, it was reported recently that nNOS also play an important role in proliferating angiogenesis in hypoxia circumstance in malignant glioma. nNOS can make the blood-brain barrier open through cGMP pathway, increase the level of VEGF, proliferate generation of new vessels; meanwhile, nitrogen monoxides can dilate the vessels and increase the blood flow to meet the need of tumor cells for oxygen. This study demonstrated that nNOS is expressed in glioma cells and the expression level of nNOS is increased with the pathological grade of glioma accordingly. Besides, this study also proved that the expression of Hif-1αand nNOS is correlated in malignant glioma, and presumed that both of them participate in promoting angiogenesis and increasing the ability of glioma in proliferation and invasion.ConclusionThe expression level of Hif-1αand nNOS grows with the increase of pathological grades. The expression of Hif-1αand nNOS are also positive correlation. Therefore, the quantitative evaluation to the expression of Hif-1αand nNOS would be an index for judging the malignance degree and the prognosis of the patients with brain glioma.更多还原