【作者】 夏熙双；

【导师】 牛光明；

【作者基本信息】 郑州大学 ， 外科学， 2010， 硕士

【摘要】 背景与目的胶质瘤是成人最常见的原发性脑肿瘤,而星形细胞瘤是最常见的神经上皮肿瘤,文献报道其发生率占颅内肿瘤的13-26%,占胶质瘤21.2-51.6%。尽管在其诊断和标准治疗,如手术、放射治疗和化疗等方面有了很大进展,这些肿瘤患者的预后依然很差。胶质瘤的治疗现状在相当程度上取决于对这些肿瘤生物学的深入理解。最近对胶质瘤的生物学研究发现酪氨酸激酶受体和信号转导通路在肿瘤发生发展中发挥重要的作用。磷酸化与去磷酸化在细胞内信息传递中起重要作用。PTEN(Phosphatase and tensin homology deleted on chromosome 10)是第一个具有磷酸酯酶活性的抑癌蛋白。PTEN负调控细胞周期及多种信号途径,抑制细胞粘附、细胞迁移、细胞分化、细胞衰老和细胞凋亡等多种生理活动,因此PTEN与肿瘤的关系备受关注,成为研究的热点。10q23染色体上PTEN肿瘤抑制基因和甲基化变异导致磷脂酰肌醇-3激酶(PI3K)信号转导通路失去控制,这已在至少60%的恶性胶质瘤中得到证实。杂合性缺失或突变引起的PTEN基因功能丧失与间变性星形细胞瘤和胶质母细胞瘤患者的生存率较低具有相关性,这表明PTEN基因对患者的预后具有重要作用。现代分子生物学认为,肿瘤的发生发展是多基因、多因素、多步骤,多阶段改变的复杂过程,单一基因改变的作用有其局限性,探讨多基因的协同作用可能会更有意义。目前PTEN基因抑癌机制还未完全研究清楚,联合检测GSK-3β、FAK在脑星形细胞瘤中表达的研究国内外文献尚未见报道。为进一步探讨PTEN基因与脑星形细胞瘤发生发展的关系,探求早期检测脑星形细胞瘤发生、分级、预后的指标及寻找抑制脑星形细胞瘤生长和侵袭的有效方法,本研究采用RT-PCR及免疫组化SP法检测了47例脑星形细胞瘤和12例非瘤脑组织中PTEN基因mRNA、GSK-3β基因mRNA、FAK蛋白的表达情况,并分析了PTEN与GSK-3β、FAK的相互关系。通过本实验研究,进一步阐明PTEN蛋白的表达在脑星形细胞瘤中的意义；阐明PTEN基因抑制脑星形细胞瘤的机制是否与上调FAK表达、下调GSK-3β表达有关,为进一步探讨脑星形细胞瘤发生发展机制及寻找抑制脑星形细胞瘤的途径提供理论基础。材料与方法收集2009年1月-2009年10月郑州大学第一、二、五附属医院神经外科手术切除、并经病理证实为星形细胞瘤的患者新鲜标本47例。所有病例均为首次发病,术前未行放疗、化疗及免疫治疗。有完整的临床病例资料。其中男性29例,女性18例,年龄19-73岁,平均39.51岁。根据WHO(2001)中枢神经系统肿瘤分类标准,对所有肿瘤进行组织学分类和分级,其中Ⅰ级7例,Ⅱ级21例,Ⅲ级14例,Ⅳ级5例。WHOⅠ-Ⅱ级星形细胞瘤合并为低度恶性组,WHOⅢ-Ⅳ级合并为高度恶性组。12例作为对照的非瘤脑组织,取自郑州市第二人民医院、郑大二附院神经外科因颅脑损伤、脑出血手术须行内减压术的患者。研究采用RT-PCR及免疫组化SP法检测了47例脑星形细胞瘤和12例非瘤脑组织中PTEN基因mRNA、GSK-3β基因mRNA、FAK蛋白的表达情况,并分析了PTEN与GSK-3β、FAK的相互关系。采用SPSS 11.0软件包处理数据,两样本率的比较采用四格表资料的χ2检验；多样本率的比较采用行列表资料的χ2检验；计量资料采用t检验；差异性分析用配对χ2检验；相关性检验用Spearman关联性分析。以α=0.05作为有统计学意义的判断标准。结果判定以P<0.05作为差异显著性检验水准。结果1.PTEN蛋白在非瘤脑组织中的阳性表达率为100%(12/12),在星形细胞瘤中的表达阳性率为53.2%(25/47),两者相比存在显著性差异(P<0.01)。PTEN蛋白在非瘤脑组织、星形细胞瘤Ⅰ级～Ⅱ级、Ⅲ级～Ⅳ级组中的表达阳性率分别为100.0%、67.9%(19/28)和31.6%(6/19)。星形细胞瘤Ⅰ级～Ⅱ级组中PTEN蛋白的表达高于星形细胞瘤Ⅲ级～Ⅳ级组,星形细胞瘤Ⅰ级～Ⅱ级与星形细胞瘤Ⅲ级～Ⅳ级间表达差异有统计学意义(P<0.05)。2.GSK-3β蛋白在非瘤脑组织中表达阳性率为66.7%(8/12),在星形细胞瘤中的表达阳性率为29.8%(14/47),两者相比存在显著性差异(P<0.05)。其蛋白在非瘤脑组织、星形细胞瘤Ⅰ级～Ⅱ级、Ⅲ级～Ⅳ级组中的表达阳性率分别为66.7%(8/12)、42.9%(12/28)和10.5%(2/19)。星形细胞瘤Ⅰ级～Ⅱ级组中GSK-3β蛋白的表达高于星形细胞瘤Ⅲ级～Ⅳ级组,星形细胞瘤Ⅰ级～Ⅱ级与星形细胞瘤Ⅲ级～Ⅳ级间表达差异有统计学意义(P<0.05)。3.FAK蛋白在非瘤脑组织中的阳性表达率为16.7%(2/12),在星形细胞瘤中的表达阳性率为53.2%(25/47),显著高于非瘤脑组织中的表达,两者相比存在显著性差异(P<0.01)。FAK蛋自在非瘤脑组织、星形细胞瘤Ⅰ级～Ⅱ级、Ⅲ级～Ⅳ级组中的表达阳性率分别为16.7%、35.7%(10/28)和78.9%(15/19)。星形细胞瘤Ⅰ级～Ⅱ级与星形细胞瘤Ⅲ级～Ⅳ级间表达差异有统计学意义(P<0.05)。4.PTEN蛋白和GSK-3β蛋白在瘤组织中表达的关系：在25例PTEN蛋白表达阳性的星型细胞瘤中GSK-3β蛋白表达阳性的有14例；在22例PTEN蛋白表达阴性的星型细胞瘤中GSK-3β蛋白表达阴性的有22例,经统计学分析两者表达呈正的相关关系(McNemar=0.628,P<0.01)。5.PTEN蛋白和FAK蛋白在瘤组织中表达的关系：在25例PTEN蛋白表达阳性的星型细胞瘤中FAK蛋白表达阳性者5例,在22例PTEN蛋白表达阴性的星型细胞瘤中FAK蛋白表达阴性者2例,经统计学分析两者表达呈负的相关关系(McNemar=-0.696, P<0.01)。结论1.PTEN蛋白在非瘤脑组织高表达,在星形细胞瘤组织中表达下调,并随星形细胞瘤病理级别的增高而表达降低,提示PTEN蛋白表达与星形细胞瘤的发生、分化、进展密切相关,检测PTEN蛋白表达有助于判断星形细胞瘤的生物学行为和恶性程度,并可以作为胶质瘤预后判断指标。2.星形细胞瘤中PTEN和FAK蛋白的异常表达呈负相关关系。提示FAK蛋白的表达上调与PTEN的表达下调在呈形细胞瘤中不是两个孤立的事件,两者可能存在某些关联,共同促进了星形细胞瘤的发生、发展。3.PTEN和GSK-3β在星形细胞瘤的发生发展进程中存在正相关性,提示可能PTEN通过GSK-3β促进肿瘤的发生发展。更多还原

【Abstract】 Backgrounds and ObjectivesGliomas are the most common primary brain tumor in adults, and astrocytomas, accounting for 13-26% in intracranial tumors and 21.2%～51.6% in gliomas, are the most frequent neuroepithelial neoplasms. But the prognosis for patients with these tumors remains poor despite advances in diagnosis and standard therapies such as surgery, radiation therapy, and chemotherapy. Progress in the treatment of gliomas now depends to a great extent on an increased understanding of the biology of these tumors. Recent insights into the biology of gliomas include the finding that tyrosine kinase receptors and signal transduction pathways play a role in tumor initiation and maintenance. Phosphorylation and dephosphorylation play an important role in intracellular signaling pathways. As the first identified tumor suppressor gene, PTEN (phosphatase and tensin homology deleted on chromosome 10) negatively regulate cell cycle and multiple signaling pathways, suppressing various physiological functions including cell adhesion, migration, differentiation, senescence, apoptosis and so on. Thus the ralationship between PTEN and tumors has attracted much attention and become a focus in medical research. Deregulation of phosphatidylin-ositol 3-kinase (PI3K) signaling pathways resulting from genetic alterations in the PTEN tumor suppressor gene on 10q23 at the level of LOH, mutation and methylation have been identified in at least 60% of glioblastoma. Loss of PTEN function by mutation or LOH correlates with poor survival in anaplastic astrocytoma and glioblastoma, suggesting that PTEN plays a role in patient outcome. In modern molecular biology, Tumor initiation and maintenance is considered to be a complex alteration process of multiple gene, multiβle factor, multiple procedure, multiple phase. The effect of single gene alteration is limited, therefore investigating the synergetic effect of polygenes may make difference. The studies nowdays has not identified clearly the tumor suppressing mechanism of PTEN, and there is no report about correlation between the expression of GSK-3β、FAK in the brain astrocytoma. In order to further explore the correlation between PTEN and the genesis and progress of astrocytoma, search markers indicating the generation, gradation and prognosis of astrocytoma, and look for usful ways to suppress the genesis and invation of astrocytoma, this research detects the expression of PTEN mRNA、GSK-3βmRNA、FAK gene and protein by immunohistochemisty in 47 cases of astrocytoma tissues and 12 cases of normal brain tisssues, and analyses the correlation between PTEN, GSK-3βand FAK. This article intends to interpret the role of PTEN protein expression in astrocytoma, investigate whether the mechanism of PTEN to suppress astrocytoma is concerned with up-regulation of FAK and down regulation of GSK-3β, and furthermore and provide certain theory basis on further detecting the mechanism of astrocytoma genesis and development and finding out the ways to suppress astrocytoma.Materials and methods47 cases of the fresh human astrocytomas tissues surgically ablated and identified by pathology were collected in the department of neurosurgery of the second affiliated hospital of Zhengzhou University from Jan,2009 to Oct,2009, including 29 cases of male and 18 cases of female,19-73 years old, mean 39.51 years of age. All cases are of intact datas and invaded for the first time. None was treated by radiotherapy, chemotherapy or immunotherapy.According to the WHO (2001) classification of central nervous system tumors, all tumors were histologically classificated and graded as 7 cases of gradeⅠ,21 cases of gradeⅡ,14 cases of gradeⅢ,5 cases of gradeⅣ. The WHO gradeⅠ-Ⅱastrocytomas merged into low-grade malignant group, and the WHO gradeⅢ-Ⅳmerged into high grade. The non-tumor brain tissues serving as control group were obtained from 12 patients requiring surgical decompression due to brain injury or cerebral hemorrhage in the neurosurgery department, in The Second People’s Hospital of Zhengzhou and The Second Affiliated Hospital of Zhengzhou University. The study used RT-PCR and immunohistochemistry SP method to detect the expression of the PTEN gene mRNA, GSK-3B gene mRNA, FAK gene and protein in 47 cases of astrocytoma and 12 cases of non-tumor brain tissue, and analyzed the correlation of PTEN and GSK-3B, FAK. The data was analysed using SPSS software (version 11.0). Chi-square statistics were used for categorical comparisons of rate of sample. The t test was used for measurement data. Spearman rank-sum correlation was used to relate the expression of markers. A value of p<0.05 was considered statistically significant.Results1. Twelve of 12 non-tumor brain tissues were positive for PTEN protein with a positive rate of 100% and twentyfive of 47 astrocytoma tissues were positive for PTEN protein with a positive rate of 53.2%. The difference of PTEN expressions was statistically significant (P<0.01) in astrocytoma and non-tumor brain tissues. The positive rates of PTEN protein expression were respectively 100.0%,67.9% and 31.6% in non-tumor brain tissue, GradeⅠ～Ⅱastrocytoma group, gradeⅢ～Ⅳastrocytoma group. The expression of PTEN protein in GradeⅠ～Ⅱgroup is higher than that of gradeⅢ～Ⅳgroup with significant difference (P<0.05).2. Eight of 12 non-tumor brain tissues were positive for GSK-3Bprotein with a positive rate of 66.7% and fourteen of 47 astrocytoma tissues were positive for GSK-3B protein with a positive rate of 29.8%. The difference of GSK-3βexpressions was statistically significant (P<0.05) in astrocytoma and non-tumor brain tissues. The positive rates of GSK-3B protein expression were respectively 66.7%,42.9% and 10.5% in non-tumor brain tissue, GradeⅠ～Ⅱastrocytoma group, gradeⅢ～Ⅳastrocytoma group. The expression of GSK-3βprotein in GradeⅠ～Ⅱgroup is higher than that of gradeⅢ～Ⅳgroup with significant difference (P<0.05).3. The positive expression rate of FAK protein was 53.2% in astrocytomas and was significantly higher than that in non-tumor brain tissue, which was 16.7%(P <0.01). The positive rates of FAK protein expression were respectively 66.7%,42.9% and 10.5% in non-tumor brain tissue, GradeⅠ～Ⅱastrocytoma group and gradeⅢ～Ⅳastrocytoma group. The differentce of FAK protein expression between GradeⅠ～Ⅱgroup and gradeⅢ～Ⅳgroup was significant (P<0.05).4. The relationship between the expression of PTEN protein and GSK-3βprotein: Of the 25 cases with positive expression of PTEN protein in astrocytomas,14 cases were detected GSK-3βprotein expression; GSK-3βprotein expression was negative in all 22 cases with negative expression of PTEN protein in the astrocytomas, positive correlation between the two protein expression was confirmed via statistical analysis (contingency coefficient r= 0.628, P<0.01).5. The relationship between the expression of PTEN protein and FAK protein: Of the 25 cases with positive expression of PTEN protein in astrocytomas,5 cases were detected FAK protein expression; FAK protein expression was negative in 2 cases out of 22 cases with negative expression of PTEN protein in the astrocytomas. The statistical analysis showed a negative correlation between the expression of the two proteins (contingency coefficient r=-0.696, P<0.01).Conclutions1. PTEN protein is highly expressed in non-tumor brain tissue and it is down regulated in astrocytoma tissues, and reducing as the pathologic grade of astrocytomas increases, suggesting that PTEN gene mutation or deletion is closely related to the occurrence, differentiation, progress of astrocytoma. Detection of PTEN protein expression will help to determine the biological behavior, malignant degree and prognosis of astrocytomas.2. There is a negative correlation between the abnormal expression of PTEN protein and that of FAK protein in astrocytoma, indicatig that the increased expression of FAK protein and the decreased expression of PTEN in astrocytic tumors are not two isolated incidents, there may be some correlation between them, which work together to promote the occurrence and development of astrocytoma.3. There is a positive correlation between PTEN and GSK-3βin the occurrence and development process of human astrocytomas, suggesting that PTEN depend on GSK-3βto promote the development of tumors.更多还原