【作者】 曲敏；

【导师】 吴安华；

【作者基本信息】 中国医科大学 ， 神经外科， 2010， 博士

【摘要】 前言胶质瘤是颅内最常见的原发恶性肿瘤,呈浸润性生长,肿瘤与正常脑组织之间无明显界限,手术无法彻底治愈,患者均死于肿瘤的复发。目前认为无法治愈的根本原因在于胶质瘤细胞的无限增殖和向周围正常脑组织的侵袭生长。研究表明,有多种生物大分子参与胶质瘤细胞的增殖和侵袭过程。这些大分子是锌依赖性的具有蛋白水解酶功能的酶蛋白分子,包括去整合素-金属蛋白酶(adisintegrin and metalloprotease, MMPs)家族、去整合素-金属蛋白酶(a disintegrin and metalloprotease, ADAMs)家族等,这些家族在结构和功能上均有一些相似之处。ADAMs家族近年来成为研究的热点。研究人员已经发现ADAM12在不同级别胶质瘤中存在表达差异,级别越高则表达越多；进一步研究发现ADAM12具有水解细胞外复合分子肝素结合性表皮生长因子(heparinbin-inding epidermal growth factor, HB-EGF)而产生活性分子表皮生长因子(epider mal growth factor, EGF)的作用。正是通过水解产生的活性分子EGF与表皮生长因子受体(epidermal growth factor receptor, EGFR)结合,使细胞外信号传递到细胞内,激活控制胶质瘤细胞增殖的相关基因完成调控作用。ADAM10作为ADAMs家族中的一员与其他成员有着相似的结构和功能。已经有研究证实ADAM10参与细胞上皮的更新,组织重建,细胞的粘连。国外研究人员研究发现ADAM10在体外胃癌细胞中有过度表达,并证实ADAM10是通过EGFR配子脱落来促进癌细胞增殖。也有人发现其能够水解细胞外Ⅳ型胶原,分析认为可能是通过水解Ⅳ型胶原促进癌细胞向远处侵袭生长。ADAM10在胶质瘤中的表达情况及是否参与癌细胞的增殖目前仍不是很清楚。本课题拟将临床不同级别胶质瘤依照世界卫生组织(world health organization,WHO)标准分为低级别和高级别两组,以脑膜瘤作为对照组,应用反转录多聚酶链式反应(reverse transcripase polymerase chain reaction, RT-PCR)对三组ADAM10基因表达进行检测,并比较表达差异。利用链霉亲合素-生物素-过氧化物酶免疫组织化学方法(StreptAvidin-BiotinComplex, SABC)检测ADAM10蛋白表达及分布,表达情况及分布进行分析,探索其可能的作用机制。本实验的目的就是为胶质瘤的靶向治疗寻找靶点并提供实验基础。材料与方法一、实验标本的获取取我院2007-2010年共计43例脑胶质瘤患者标本,低级别胶质瘤22例,高级别胶质瘤21例,取脑膜瘤10例作为对照组。所有标本均于肿瘤中心采取,立即置于-70℃保存,并保留病例信息。所有标本均经过病理学核实。二、实验方法(一)肿瘤组织RNA的提取按照TaKaRa公司说明利用RNAiso Plus从组织标本中提取RNA。取100mg-70℃保存的瘤组织剪成匀浆状,立即放入1.5ml去RNA酶的EP管中加入1mlRNAiso Plus,室温静置5分钟,取上清加入0.2ml氯仿,静置后离心,取上清后加入异丙醇静置后再离心可见白色片状物,干燥后加入40ul去RNA酶水溶解用紫外分光光度仪测RNA纯度及含量。(二)两步法RT-PCR：引物序列参考文献由TaKaRa公司合成,上下游序列分别为5’-TGGGTCAAAAAGAAAATGGC-3’和5’-CCCAGGTTTCAGTTTGCATT-3’。扩增片段长度为281bp,采用β-actin为内参对照,上游：5’-AAATCGTGCGTGACATTAA-3’,下游：5’-CTCG TCATACTCCTGCCTG-3’扩增片段大小为474 bp。RT体系为10ul,取2.5ul模板(cDNA)进行扩增。PCR体系在94℃下变性2分钟,循环条件为94℃30秒,55℃30秒,72℃2分钟,共30个循环,最后延伸10分钟。取产物8ul通过电泳利用琼脂糖凝胶进行分离,并在紫外线灯下照相。最后通过凝胶灰度分析仪进行统计分析。(三)免疫组织化学染色按照SABC染色试剂盒说明进行操作,结果判断：在光镜下有棕黄色颗粒反应出现在细胞膜、细胞质均为阳性反应细胞,于5个不同高倍视野相同范围下计数500个细胞及免疫组化染色阳性细胞数,阳性细胞率<5%记为(-),5%-50%为(+),50%-75%为(++),>75%为(+++)。统计学处理。利用SPSS14.0软件对结果进行数据分析。不同组间参数比较利用One-wayANOVA分析,p<0.05具有统计学意义。结果一、ADAM10基因mRNA的表达对三组共63个条带灰度扫描,平均灰度值提示高级别组ADAM10基因表达最多,对照组略有表达。平均灰度单因素方差分析提示高级别组和低级别组比较具有显著性差异(t=13.31,p<0.05)；低级别组与对照组比较差异明显(t=22.83,p<0.01)。二、ADAM10蛋白的表达对照组高倍镜下可见47个阳性细胞(9.4%),低级别组可见269个阳性细胞(53.8%),高级别组可见382个阳性细胞(76.4%)。ADAM10蛋白免疫组化染色显示对照组中可以看到稀少阳性染色细胞；低级别组中可以看到很多瘤细胞结构已经破坏,呈空泡状,背景呈现非特异染色。可以看到散在的瘤细胞胞膜上和胞质部分阳性染色；高级组上可以看到较多阳性染色的瘤细胞,肿瘤内血管壁上有环形阳性染色。结论1、ADAM10在脑膜瘤和胶质瘤中均有表达,表达量与胶质瘤级别呈正相关性。胶质瘤中的表达与脑膜瘤中表达的差异有统计学意义。2、ADAM10在胶质瘤血管壁上有明显的表达,其可能是胶质瘤血管壁通透性增加引起瘤周水肿的机制之一。更多还原

【Abstract】 Intracranial glioma is the most common primary malignant tumor,which is characterized as invasive growth.Because there are no obvious boundaries between tumor and normal brain tissue so surgery can not be completely cured.And all the patients with giloma died of tumor recurrence finally. The tumor in general bear a grim cure due to diffuse infiltration of tumor cells into the surrounding brain tissue, preventing complete surgical removal of the tumor. Studies have shown that a variety of biological macromolecules involved in proliferation and invasion of glioma cells in the process of growth. These molecules are zinc-dependent protein with proteolytic enzymes function,including MMPs family and ADAMs family.There are some similarities with functions and structures in the two families. ADAMs family have caused widely concern in recent years,and some researchers have found that there were some differents among the ADAM12 expression in different grades of giloma.The higher level of grades,the higher level of expression.Further study have shown that ADAM12 can produce EGF by hydrolysising HB-EGF,which involving the signaling process.Then EGF conbined with EGFR so that the extracellular signal delivered to cell to activate the genes associated with the proliferation of glioma cells. Some studies have proven that AD AM10 involved the epithelial update of cells,the tissue reconstruction,and cell adhesion.Foreign researchers have found that ADAM10 was overexpressed in gastric cells in vitro and confirmed that ADAM10 promotes cancer cells proliferation by shedding gametes. It was also found to be hydrolysis of extracellular collagen typeⅣ, which was thought to promote the growth of cancer cell invasion to the distance.In our study we divided the sample into two groups of low-level and high-level according to the WHO standards with meningioma as the control group.The three groups gene are detected through RT-PCR and the results were compared with each other. We applied immunohistochemistry to show the protein of ADAM10 in the cell to explore the mechanism.All we done in the experiments are to find the the target for targeted therapy and provide the experimental basis.Materials and Methods1. Obtain experimental specimens43casesof giloma samples are collected in our hospital from 2007-2010,including 22 cases of low-grade and 21 cases of high-grade.10 cases of meningioma are served as control group.The samples were from the core of the tumor and were saved at-70℃immediately.The information of the patients are collected and the pathological results were confirmed.2. Experimental Methods(1) Extract RNAAccording to the TaKaRa description we extract the RNA from the tumor samples.The 100mg tumor tissue were made into homogenates and was put into 1.5ml free RNA enzymes EP tube and was mingled with 1ml RNAiso Plus. Put it aside for 5 minutes at room temperature. Add 0.2ml chloroform to Supernatant obtained, put it aside, after centrifugation, then the supernatant was added to isopropyl alcohol, white flakes can be seen after centrifugation and then put it aside, adding 40ul water-soluble enzymes to the RNA after dried,then RNA purity and content was measured by UV spectrophotometer.(2) Two-step RT-PCR:Reference primers synthesized by TaKaRa Company, upstream and downstream sequences:5’-TGGGTCAAAAAGAAAATGGC-3’和5’-CCCAGGTTTCAGTTTGCA TT-3’.Amplified fragment length is 281bp, usingβ-actin as an internal control. upstream and downstream respective is as follw:5’-AAATCGTGCGTGACATTAA-3’ ,5’-CTCGTCATACTCCTGCCTG-3’, the size of amplify fragment is 474bp,the whole system is 10ul,take 2.5ul template (cDNA)to amplify,RT system denature at 94℃for 2 minutes,cycle condition is at 94℃for 30secend, at 55℃for 30 secend,at 72℃for 2 minute,add up to 30 cycles,extension for 10 minute. The products were separated through electrophoresis using agarose gel,photographs were taken in the UV lamp camera,statistical analysis was made by gray gel analyzer. (3) ImmunohistochemistryOperation is made according to instructions of SABC staining kit.In the light microscope, there are brown-yellow granules reaction appears on the cell membrane,in the cytoplasm,which was regarded as positive reaction.500 cells were counted in 5 different high power field and the rate of positive cell less than 5% is marked(-),5%-50% marked (+),50%-75% marked (++),>75% marked (+++)(4) StatisticsThe results of data was analysised using SPSS 14.0 software. Different groups parameters were compared using One-way ANOVA analysis, p<0.05 means significant statistically.Results1. The expression of AD AM10 gene63 grayscale strip was scaned and the results showed that the most expression was in high-grade group, little expression was in control group.Single-factor analysis of the average gray showed that the different between high-grade group and low-grade group was statistically significant (f=13.31, p<0.05), Low-grade group and control group differences are significant (t=22.83, p<0.01)2. The expression of AD AM10 proteinThere are 47 positive cells in the control group in the high-powered microscope(9.4%),269 postive cells in the low-grade group(53.8%),382 positive cells in the:high-grade group(76.4%).Many tumor cells had been destroied in the low-grade,tumor cell membrane and cytoplasm was sporadic positive staining. circular positive staining was showed in the blood vessel walls.Conclusion1. ADAM 10 is expressed in meningiomas and gliomas and the expression was positively correlated with the level of glioma. Glioma and meningioma was statistically significant differences in the expression.2. ADAM 10 expression are shown in the vassel of the glioma,which may be one of the mechanisms that increased permeability of glioma blood vessel wall that caused peritumoral edema.更多还原