【作者】 韩晞；

【导师】 卢亦成；

【作者基本信息】 第二军医大学 ， 外科学（神经外科）， 2001， 博士

【摘要】 胶质瘤是中枢神经系统最常见的原发性恶性肿瘤，其侵袭性生长的特点给手 术彻底切除带来困难，是胶质瘤复发率高、预后不良的重要原因。因此，肿瘤侵 袭生长机制成为近年来胶质瘤的研究方向之一。 以往的研究表明，胶质瘤细胞的侵袭性与细胞外基质密切相关。在神经发育 过程中，神经系统的细胞外基质趋于液态，有利于神经细胞的移动和轴突延伸。 成年后，细胞外基质变得不可溶，细胞的移动受到限制，使脑组织的形态和功能 得以维持。在胶质瘤形成过程中，细胞外基质发生类似神经发育中的改变，脑内 微环境有利于肿瘤细胞的迁移和侵袭。蛋白聚糖作为神经系统细胞外基质的主要 成分，其表达调节与胶质瘤生长密切相关。 短蛋白聚糖（brevican／Brain Enriched HyAluronan Binding，BEHAB，脑富含 的透明质酸结合蛋白）是1994年发现的一种硫酸软骨素蛋白聚糖（chondroitin sulfate proteoglycan，CSPG），属于凝集素蛋白聚糖（lectican）家族，是脑内最富 含的细胞外基质分子之一。Brevican存在分泌型和糖基磷脂酰肌醇（glycosyl－ phosphatidylinositol，GPI）锚定型两种，由胶质细胞和神经元合成，具有高度的 组织特异性，其表达只限于中枢神经系统，而周围组织中未见表达。brevican的 表达在胚胎晚期开始出现，随神经发育过程而显著增高，成年以后仅维持低水平 的表达。在中枢神经系统损伤时，brevican表达有明显上调。令人感兴趣的是， brevican在脑胶质瘤中也有明显增高。体内外研究提示，brevican在胶质瘤中的表 达需要脑内环境因素，并与胶质瘤侵袭性有关。但是胶质瘤中brevican是否由肿 瘤细胞表达尚无组织学证据，brevican表达量与胶质瘤恶性程度的关系亦未见报 道，brevican在胶质瘤及其侵袭现象中的作用及其机制仍不清楚。 本课题采用RT-PCR方法，从人脑星形细胞瘤中克隆了人分泌型brevican的 全长基因，为进行brevican蛋白表达、抗体制备及生物活性研究建立基础；并且 采用半定量RT-PCR方法结合原位杂交组织学观察，检测了brevican在脑胶质瘤 和非胶质来源脑肿瘤中的表达。 二 中文摘要二 主要结果： 一、人分泌型 breian cDNA的克隆： 手术中取新鲜脑胶质瘤标本，置液氮中速冻，异硫氰酸肌一步法提取总RNA。 以寡聚dT为引物对polyw“RNA进行逆转录，合成cDNA第一链JCR扩增p－actin 467习24片段，检测逆转录反应的效率。 根据GeneBank的检索结果，设计两对引物：引物EHI／EHZ扩增brevim 48－1637，引物SHI／SHZ扩增br的can 1605－2812。PCR分别得到长约1．6讪的片段 EH－PCR和长约 1．Zkb的片段 SH－PCR。利用两者重叠区中的限制性内切酶 Hnd Ill 位点及引物设计时引入的限制性内切酶EC。RI和Sal位点，将两段PCR产物分 别插人 pBlues吟t SR＋质粒的多克隆位点中，经再次酶切、连接，最终得到包含 完整阅读框的 b…an 48毛812。其中有两处点突变：严突变为 C，相应的编码氨 基酸仍为亮氨酸；A’“突变为T，相应的编码氨基酸由谷氨酸变为缀氨酸。 二、brevican M在人脑胶质瘤中的表达： 以地高辛标记的 brevican 1605毛 cRNA探针进行原位杂交。光镜下观察 brevican阳性细胞胞浆呈蓝紫色，大小不一，不均匀散在分布，密度明显大于正 常脑组织，显色适度的细胞中可见略呈浅染的异型细胞核，表明为肿瘤细胞。结 果显示所有23例胶质瘤标本均有明显的阳性反应（100％），其中一例小脑髓母细 胞瘤中可见肿瘤浸润部位呈强阳性反应，而邻近的正常皮层则无阳性细胞：对照 组7例非胶质源性脑肿瘤和脑转移瘤均未见到阳性反应。提示brevican rnRNA是 由胶质瘤细胞表达的，并且具有高度的特异性，这是目前发现的脑肿瘤相关基因 蛋白中特异性最高的。 采用半定量RTPCR方法，以pdctin为内参照检测brevican tnRNA在脑肿瘤 中的表达。结果显示23例胶质瘤中brevican M阳性率为91，3％Q1乃3人 平更多还原

【Abstract】 Glioma is the most common among the malignant primary tumors in the central nervous system. The highly invasive behavior of giloma, which makes it particularly difficult to completely remove them by surgery, is an important cause for the high rate of recurrence. More and more studies focus on the mechanism of invasion of glioma in recent years. Former data showed that the extracelluar matrix (ECM) plays important roles in the aggressive invasion of glioma. During brain development, the neural ECM tends to be more liquid and is permissive for the moving of neural cells and the extension of axons. In adult brain, ECM becomes more insoluble and the moving of cells is restricted to maintain the morphology and function of brain. Changes of ECM in glioina are similar to that in brain development, which make it easier for tumor cells to move and invasion. Regulation of chondroitin sulfate proteoglycans (CSPGs) as the main elements of ECM in the nervous system is correlated with glioma. Brevican/BEHAB (Brain Enriched Hyaluronan Binding), which was found in 1994, is a new member of the lectican family of CSPGs. It is one of the most enriched extracelluar matrix molecules in brain. Brevican has both secreted and glycisyphosphatidylinsitol (GP1)- anchored isoforms and is expressed by neurons and glial cells. The expression of brevican is remarkably restricted to the CNS and can’S be found in peripheral tissues. First detected at embryonic days, brevican increases with neural development and maintains low levels of expression in adult brain. In brain injury, the expression of brevican is upgraded. Interestingly, brevican is increased in glioma. Both in tow and in mtm studies show that the expression of brevican in glioma needs intracranial brain environment and correlates with glioma invasion. The roles of brevican in giloma and its invasive growth are not completely understood now. In this study, we cloned the full cDNA of human brevican secreted isoform from a human astrocytoma by RT-PCR and detected the levels of brevican mRNA in glioma and nonglial brain tumors by in situ hybridization and semiquantitative RT-PCR. 5 The results are as follows: 1. cDNA cloning of human brevican secreted isoform. Surgical sample of a human astrocytoma was fresh-frozen in liquid nitrogen. Total RNA was prepared by guandimium isothiocyanate. First strand cDNA was synthesized by RT using oligo-dT. A fragment of [3-actin 476-924 was amplified by PCR to test the quality of cDNA. Two pairs of primers were designed according to secreted brevican mRNA from GeneBank search results: EHI/EH2 (corresponding to brevican 48-1637) and SHI/SH2 (corresponding to brevican 1605-2812). A 1.6kb fragment (EH-PCR) and another 1.2kb fragment (SH-PCR) were expected, with a unique Hind III restriction site existed in the overlapping part. The PCR products were cloned into pBluescnpt SK+ vectors and then ligated into a complete one, which included the whole open reading frame of brevican and was inserted into pBluescript SK+ vector between EcoR I and Sal I sites. Tow point mutations were found: T~ to C and A to T. Expression of brevican mRNA in human glioma. In situ hybridization was carried with a digoxin-labelled brevican cRNA probe (corresponding to brevican 1605-2812). Under microscope, positive tumor cells with blue更多还原