【作者】 苗红；

【导师】 宋福林；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2010， 硕士

【摘要】 背景与目的：脑星形细胞瘤是来源于神经上皮组织的常见的颅内肿瘤,主要为肿瘤性星形细胞所构成。肿瘤级别和恶性程度的确定对治疗方案的选择有重要的价值。以往对星形细胞瘤的诊断主要依据磁共振平扫和增强检查,但对星形细胞瘤恶性度分级存在局限性。MR波谱(MR spectroscopy,MRS)是一种利用MR现象和化学位移作用对一系列特定原子核及其化合物进行分析的方法,是目前研究人体器官、组织代谢、生化改变及化合物定量分析的惟一无损伤性方法。可以获得活体组织的生化代谢信息。星形细胞瘤由于其组织学形态复杂,病理分级繁琐,免疫表型差异较大,故其预后差异显著,难以判断,近年来研究表明,些基因改变和物质的表达和星形细胞瘤增殖活性、病理级别及患者预后密切相关。Ki-67是目前已知的增殖抗原中最具增殖能力的代表指标。bcl-2是目前研究较为深入的凋亡抑制基因。为进一步明确星形细胞瘤预后影响因素、指导临床治疗,本文目的：1、研究脑星形细胞瘤氢质子磁共振波谱(proton magnetic resonance spectroscopy 1H-MRS)表现及其临床意义；2、探讨脑星形细胞瘤的1H-MRS特点与其病理级别相关性。3、探讨Ki-67和bcl-2在脑星形细胞瘤中的表达及其与病理级别的相关性。4、探讨脑星形细胞瘤磁共振波谱与肿瘤细胞的bcl-2蛋白、细胞核增殖抗原(Ki-67)蛋白表达的相关性。材料及方法：1.材料搜集2006年1月至2009年12月沈阳军区总医院经临床手术及病理证实的脑星形细胞瘤52例,年龄10-77岁,平均38岁,男28例,女24例。所有患者均行常规MR和1H-MRS检查。所有标本经中性4%福尔马林液固定,石蜡包埋,选择典型病灶的石蜡块连续切片。2.方法2.1 MR设备及成像方法：磁共振检查采用GE Signa MR／iTM1.5T Hispeed Plus超导型磁共振扫描仪。用标准头线圈作发射和接收线圈。患者取仰卧位,所有患者均行常规T1加权像、T2加权像、增强扫描和多体素质子波谱扫描。常规增强T1WI,静脉团注GD-DTPA,剂量：0.2mmol／kg体重.在增强扫描前行多体素波谱扫描,点分辨波谱(PRESS)序列,对肿瘤区、对侧正常脑组织区进行测量。检测不同区域代谢物变化。2.2免疫组化方法：应用免疫组化染色技术检测正常脑组织、星形细胞瘤中Ki-67和bcl-2蛋白的表达情况。分析肿瘤实质内NAA／Cho、Cho／Cr和NAA／Cr值与肿瘤级别的关系,并分别与Ki-67、bcl-2进行相关性分析。采用链霉菌抗生物素蛋白一氧化物酶连接法(Streptavidin／Peroxidase,SP),一抗为鼠抗人bcl-2单克隆抗体(即用型)；鼠抗人Ki-67单克隆抗体(即用型)均购自北京中山生物技术有限公司。石蜡切片常规脱蜡、水化,选择典型病灶的石蜡块连续切片。继以免疫组化试剂盒进行检测,操作步骤按说明书进行。3.结果判定3.1免疫组化判定标准：bcl-2阳性细胞以细胞浆出现棕黄色颗粒为标准,Ki-67以细胞核内出现棕黄色颗粒为阳性细胞,根据阳性细胞所占百分比和染色强度分为3级：阳性细胞数≥25%,着浅色棕色为(+)；阳性细胞数≥75%,着深棕色为(+++)；阳性细胞数及显色强度介于二者之间为(++)；3.2统计分析：数据统计应用SPSS 11.5软件,采用χ2检验、t检验和Spearman等级相关分析。结果：1.脑星形细胞瘤的MRI及’H-MRS表现：T1WI、T2WI多表现为均匀或不均匀信号,增强扫描为明显及部分强化。肌酸(Cr)轻度下降,N-乙酰天门冬氨酸(NAA)显著下降,胆碱(Cho)显著增高。NAA／Cho. NAA／Cr比值下降,Cho／Cr比值升高,高级别脑星形细胞瘤的Cho／Cr高于低级别星形细胞瘤和对照组,NAA／Cho.NAA／Cr低于低级别星形细胞瘤和对照组。脑星形细胞瘤的NAA／Cho.Cho／Cr.NAA／Cr比值与病理级别相关,NAA／Cho.N AA／Cr比值存在负相关关系(相关系数rs分别为-0.632,-0.798),Cho／Cr比值存在正相关关系(相关系数rs为0.816)。2.免疫组化结果：Ki-67和bcl-2在星形细胞瘤中有阳性表达,且随着脑星形细胞瘤病理分级程度的升高而升高(r=0.600,P<0.01)；Ki-67和bcl-2在低级别和高级别组间差异显著(P<0.05)；Ki-67和bcl-2表达密切相关(P<0.05)。星形细胞瘤Cho／Cr与bcl-2蛋白表达呈明显正相关(r=0.745,p<0.01)；星形细胞瘤NAA／Cho与bcl-2蛋白表达呈明显负相关(r=-0.253,p<0.01)。星形细胞瘤Cho／Cr与Ki-67蛋白表达呈明显正相关(r=0.818,p<0.01),星形细胞瘤NAA／Cho与Ki-67蛋白表达呈明显负相关(r=-0.501,p<0.01),星形细胞瘤NAA／Cr与bcl-2蛋白表达呈负相关(r=-0.374,p<0.01),星形细胞瘤NAA／Cr与Ki-67呈负相关(r=-0.436,p<0.01)。结论：1、Ki-67在低级别和高级别脑星形细胞瘤中有不同程度的表达,且表达程度随级别的增高而增加。2、bcl-2在低级别和高级别脑星形细胞瘤中有不同程度的表达,且表达程度随级别的增高而增加。3、bcl-2和Ki-67在脑星形细胞瘤的发生、发展中起重要作用,者联合检测可作为判断脑星形细胞瘤恶性程度的指标。4.’H-MRS可评价脑心形细胞瘤的分级,MRS基本反映着肿瘤细胞ki-67及bcl-2表达情况,可在术前预测脑星形细胞瘤代谢特性,肿瘤生长潜能,侵袭能力和预后。其中NAA／Cho和Cho／Cr比值反映肿瘤级别较稳定,较NAA/Cr更有意义。更多还原

【Abstract】 Background and Objective:Astrocytoma is a common intracranial neoplasms derived from the neural epithelium and mainly composed of neoplastic astrocytes. Definitions of tumor hierarchy and the degree of malignancy carry the significant values in determining treatment programs. In the past, the diagnosis of astrocytoma was based primarily on plain and enhanced MRI examinations,which,however, had the limitations in the grading of malignant astrocytoma. MR spectroscopy (MR spectroscopy, MRS) is not only used to analyze a series of specific nucleus and its compounds by MR phenomenon and the role of chemical shift,but also is the only non-injury approach of studying human organs, tissue metabolism, biochemical changes and quantitative analysis of compounds, acquiring biochemical metabolism information of living tissue.With respect to complicated histologic, pathological grade and obvious disparity in immunophenotypic features,their prognoses are quite different and difficult to evaluate. Recent studies suggest that some gene changes,material expression, glioma proliferation and pathological grade are closely related with patient prognosis. Ki-67 proliferation antigen is the most known representative of proliferation index. bcl-2 is a more in-depth study of apoptosis suppressor gene.To make clear the influencing factors of astrocytoma prognosis, guide clinical treatment, purposes of this paper are as follows:1.to study the finding of astrocytoma proton magnetic resonance spectroscopy (proton magnetic resonance spectroscopy’H-MRS), its clinical significance and the ’H-MRS features associated with pathological levels; 2.to investigate the relationship between the Ki-67,Bcl-2 expressions of human astrocytoma. and its pathological classification；3.to approach the correlation between magnetic resonance spectroscopy of astrocytic tumors and the expressions of tumor cells bcl-2 protein, proliferating cell nuclear antigen (Ki-67).Materials and Methods:1. Materials:Collected from January 2006 to December 2009 by the General Hospital of Shenyang Military Region,52 cases of astrocytoma including 28 males and 24 females were confirmed by clinical surgery and pathology,Their ages were from 10to77 years, and the average was 38 years,. All patients underwent conventional MR and 1H-MRS examinations. All specimens were fixed by formalin, embedded by paraffin,and the typical lesions of paraffin blocks were serially cut.2. Method:2.1 MR equipment and imagingMagnetic resonance imaging was conducted by GE Signa MR/iTMl.5T Hispeed Plus superconducting magnetic resonance scanner. Standard head coil for transmitting and receiving coils. all patients were Supine position and underwent conventional T1-weighted imaging, T2-weighted images, enhanced scanning and multi-voxel spectroscopy scan. Intravenous bolus injection dose of GD-DTPA is 0.2mmol/kg weight by conventional contrast enhanced T2WI. before enhancing, the tumor area and the contralateral normal brain tissue area were measured by multi-voxel spectroscopy scanning and point-resolved spectroscopy (PRESS) sequence. Metabolite changes were detected in different regions.2.2 ImmunohistochemistryThe expressions of Ki-67 and bcl-2 protein in normal brain tissue astrocytoma were detected by applications of tissue microarray and immunohistochemical staining. The maximum of NAA/Cho, Cho/cr and the minimum of NAA/cr were recorded and the relationship with tumor grade was analyzed. The relationships between the above indices and ki-67, bcl-2 were investigated respectively.By streptavidin protein monoxide enzyme-linked method (Streptavidin/Peroxidase, SP), an antibody is mouse anti-human bcl-2 monoclonal antibody (to-use type); the other is mouse anti-human Ki-67 monoclonal antibody (to-use type) and both were purchased from Beijing Zhongshan Biotechnology Co., Ltd.. Paraffin sections of conventional de waxing, hydration, select the typical lesions of paraffin blocks of serial sections. Followed by immunohistochemistry kit for detection,the steps according to a prospectus.3. The results determineThe bcl-2 positive cells appeared brown-yellow granules in cytoplasm and Ki-67 positive cells appeared it in nucleus as a standard. According to the percentage of positive cells and staining intensity,these cells were divided into three:the number of positive cells≥25%, with light brown for the (+); positive cells>75%, with dark brown positive cells for the (+++); and color intensity in between for the (++);Results:1. The findings of MRI and’H-MRS:MRI findings were as followings:the signal intensity was mostly heterogeneous on T1WI,T2WI,the apparent and partial intensified.Cr slightly decreased, and NAA distinctly decreased, but Cho obviously increased in astrocytoma. The ratio of NAA/Cho、NAA/Cr decreased,howerer, Cho/Cr increased. The ratio of Cho/cr in the high grade astroaytoma was higher, while NAA/cr and NAA/Cho was lower than that of low grade astrocytoma and control group. There was significant correlation between the ratios of NAA/Cr, Cho/cr and NAA/Cho and the pathological grading of astrocytoma,there was significantly negative correlation in the ratios of NAA/Cr and NAA/Cho (rs was-0.632,-0.798 respectively),and positive correlation in the ratios of Cho/Cr(rs was 0.816).2. Immunohistochemistry results:The protein expressions of ki-67 and bcl-2 in astrocytoma was related with malignancy of astrocytoma. Positve expression of ki-67、bcl-2 increased with increasing clinicopathologic grades (r=0.600, p<0.01). Ki-67 and bcl-2 in the low-level and high-level group were significantly different (P<0.05); The expressions of Ki-67 and bcl-2 was closely related (P<0.05).The ratio of Cho/cr was strongly positively correlated with expressions of bcl-2 and ki-67(r=0.745, p<0.01; r=0.818, pp<0.01 respectively).The ratio of NAA/Cho was weakly positively correlated with expressions of bcl-2 and ki-67(r=-0.253, p<0.01; r=-0.501, p<0.01 respectively). The NAA/cr of astrocytoma was weakly related with expressions of bcl-2 (r=-0.374, p<0.01) and ki-67 (r=-0.436, p<0.01).Conclusion1. The degree of expressions of Ki-67 in low grade and high grade astrocytoma was distinct and increased with the level.2. The degree of expressions of bcl-2 in low grade and high grade astrocytoma was different and raised with the hierarchy3. bcl-2 and Ki-67 played significant roles in the occurrence and development of cerebral astrocytoma,, and the two joint detections can be used to judge the degree of malignant astrocytoma indicators.4.’H-MRS has made it possible to evaluate the grade of astrocytoma. MRS can reflect the protein expressions of Ki-67 and bcl-2 in astrocytoma in principle, predict metabolism, the potential growth, invasiveness of astrocytoma and also can forecast prognosis of astrocytomas before surgery. The ratios of Cho/cr, NAA/Cho reflected pathologic grade of astrocytoma more steadily and were more valuable than NAA/cr.更多还原