INPP4B和PTEN在人脑胶质瘤中的表达及意义

发布时间：2018-05-07 05:33

本文选题：胶质瘤 + INPP4B　；参考：《川北医学院》2017年硕士论文

【摘要】：背景及目的:人脑胶质瘤多起源于神经胶质细胞,属于神经上皮源性肿瘤,约占中枢神经系统中所有肿瘤的27%;在颅内原发性恶性肿瘤中胶质瘤是最常见的,约占80%。胶质母细胞瘤(Glioblastoma,GBM,WHOⅣ级)在整个中枢神经系统原发性恶性肿瘤中,发病率是最高的,占到46%左右,男性发病率高于女性。胶质瘤具有起病隐匿,生长迅速,恶性程度相对较高,无明显边界,且呈浸润性生长等特点,目前治疗效果总体欠佳,在颅内原发性肿瘤中,胶质瘤患者的病死率是最高的,仅有1年左右的中位生存时间。与所有恶性肿瘤一样,胶质瘤的发生发展是一个多步骤、多因素的过程,但目前确切的发生机制仍有待进一步研究;伴随着科学技术的进步,与胶质瘤发生发展密切相关的肿瘤抑制因子越来越多的被发现,这对于寻找针对胶质瘤的靶基因或靶蛋白,以及胶质瘤诊断分级分型、开发分子靶向药物和判断预后等具有极大的推动作用。在目前的研究基础上,已发现以下3条基因信号通路的异常可能是胶质瘤发生发展的部分因素,包括:EGFR/PTEN/PI3K途径,Rb-E2F/CDK4、6/P16-cyclin D途径,P53/MDM2/P12、P21途径。INPP4B基因是定位于人染色体4q31.21的一个潜在肿瘤抑制基因,其编码的产物为磷脂酰肌醇4-磷酸酶Ⅱ型(INPP4B,inositol polyphosphate4-phosphatase type II),是一种脂质磷酸酶。根据已有的研究结果显示,作为一个潜在的肿瘤抑制因子,在许多恶性肿瘤中存在INPP4B的异常表达,如在白血病、乳腺癌、前列腺癌、黑色素瘤、肝癌、胃癌等恶性肿瘤中均发现INPP4B的表达缺失或表达异常。INPP4B主要使磷脂酰肌醇3,4-二磷酸(PI(3,4)P2)的D4位基团去磷酸化,其产物为3-磷酸磷脂酰肌醇(PI(3)P);而PI(3,4)P2能使AKT激活,当PI(3,4)P2降低时,则PI3K/AKT信号传导通路作用被抑制,从而INPP4B起到抑制肿瘤的发生。人第10号染色体缺失的磷酸酶和张力蛋白同源物基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)是一个定位于染色体10q23.31,编码由403个氨基酸组成的具有磷脂和蛋白质的双重磷脂酶活性的蛋白。PTEN作为目前在肿瘤研究中较为火热的抑癌基因之一,其在细胞周期的调控、诱导细胞的凋亡以及细胞的迁移粘附等方面具有重要的调控作用。PTEN蛋白主要通过去磷酸化作用,使磷脂酰肌醇3,4,5-三磷酸(PI(3,4,5)P3)的D3位基团脱磷酸,来调节细胞内PI(3,4,5)P3水平,对PI3K/AKT途径进行负调控。本实验拟通过检测INPP4B与PTEN在人脑胶质瘤中的表达情况及二者的相关性,以期能在分子水平对胶质瘤发生发展机制作初步探讨,同时希望能对胶质瘤的诊断分级、预后判断及治疗等方面提供理论依据。方法:收集川北医学院附属医院神经外科2015年1月到2016年8月胶质瘤手术切除并且病理明确诊断的标本60例,其中男性患者33例,女性患者27例,Ⅰ级胶质瘤5例,Ⅱ级胶质瘤26例,Ⅲ级胶质瘤20例,Ⅳ级胶质瘤9例。以上胶质瘤标本均按照2007年WHO的中枢神经系统肿瘤分类分级来进行病理分类分级,所收集标本均有完整病历资料,且术前未进行放射治疗、化学治疗等抗肿瘤治疗。另外再收集正常脑组织标本25例作为对照分析(标本主要来源于脑外伤等高颅内压患者手术时切除的挫伤坏死脑组织)。所有标本切下后,立即予以4℃左右PBS液反复冲洗去除血液,清除坏死、电灼失活的组织,然后将标本一分为二,一份置于4%的多聚甲醛溶液中固定1到2日后石蜡包埋备用,另一份放于液氮中转移至实验室,然后置于-80℃冰箱中保存备用。采用免疫组织化学法(IHC)和实时荧光定量PCR(qRT-PCR)法分别测定正常脑组织和胶质瘤中INPP4B和PTEN的表达情况;并采用统计软件SPSS22.0对实验数据进行统计分析。结果:1免疫组化实验结果显示:INPP4B在正常脑组织组和低级别胶质瘤组、高级别胶质瘤组三组间表达差异有统计学意义(P0.05);其阳性率:正常脑组织组中为92%,低级别胶质瘤组中为67.7%,高级别胶质瘤组中为37.9%;低级别胶质瘤组中INPP4B表达高于高级别胶质瘤组(P0.05),正常脑组织组中INPP4B表达高于低级别胶质瘤组、高级别胶质瘤组(P0.05)。PTEN在正常脑组织组和低级别胶质瘤组、高级别胶质瘤组三组间表达差异有统计学意义(P0.05);其阳性率:正常脑组织组中为96%,低级别胶质瘤组中为74.2%,高级别胶质瘤组中为41.4%;低级别胶质瘤组中PTEN表达高于高级别胶质瘤组(P0.05),正常脑组织中PTEN表达高于低级别胶质瘤组、高级别胶质瘤组(P0.05)。2 qRT-PCR实验结果显示:INPP4B mRNA在正常脑组织组和低级别胶质瘤组、高级别胶质瘤组三组间表达差异有统计学意义(P0.05),低级别胶质瘤组中INPP4B mRNA的表达水平低于正常脑组织组(P0.05),高级别胶质瘤组中INPP4B mRNA的表达水平低于低级别胶质瘤组与正常脑组织组(P0.05);PTEN mRNA在正常脑组织组和低级别胶质瘤组、高级别胶质瘤组三组间表达差异有统计学意义(P0.05),低级别胶质瘤组中PTEN mRNA的表达水平低于正常脑组织组(P0.05),高级别胶质瘤组中PTEN mRNA的表达水平低于低级别胶质瘤组与正常脑组织组(P0.05);INPP4B与PTEN在正常脑组织、低级别胶质瘤、高级别胶质瘤三组中表达成正相关,(r=0.752,P=0.000)。结论:1 INPP4B与PTEN的表达水平均与胶质瘤的恶性级别呈负相关,随着胶质瘤恶性程度的增高二者的表达水平降低,提示二者可能为胶质瘤的抑制基因。2 INPP4B与PTEN在胶质瘤中的表达水平具有正相关性,推测其可能在抑制胶质瘤的发生、发展、侵袭增殖等方面具有协同作用。3基于INPP4B与PTEN在胶质瘤中的表达具有相同的下降趋势,因此推断联合检测INPP4B与PTEN可能作为胶质瘤诊断分级、预后判断等方面的参考指标。
[Abstract]:Background and objective: glioma mostly originates from neuroglial cells and belongs to neuroepithelial neoplasm, which accounts for about 27% of all tumors in the central nervous system, and gliomas are the most common in primary intracranial malignant tumors, accounting for the primary malignant swelling of the 80%. glioblastoma (Glioblastoma, GBM, WHO IV) in the whole central nervous system. In the tumor, the incidence is the highest, accounting for about 46%. The incidence of male is higher than that of the female. Glioma has the characteristics of insidious onset, rapid growth, relatively high malignancy, no obvious boundary, and infiltrative growth, and the overall treatment effect is not good at present. In the primary intracranial tumors, the mortality of glioma patients is the highest, only 1 years left. Right median survival time. Like all malignant tumors, the development of glioma is a multistep, multi factor process, but the exact mechanism remains to be further studied. With the progress of science and technology, more and more tumor suppressor factors, closely related to the development of glioma, are found. The target gene or target protein of glioma, as well as the diagnosis and classification of glioma, the development of molecular targeting drugs and prognosis have great impetus. On the basis of current research, the following 3 gene signal pathways have been found to be part of the development of glioma, including: EGFR/PTEN/PI3K pathway, Rb-E2F/ CDK4,6/P16-cyclin D pathway, P53/MDM2/P12, P21 pathway.INPP4B gene is a potential tumor suppressor gene located on human chromosome 4q31.21, and its encoding product is phosphatidyl inositol 4- phosphatase type II (INPP4B, inositol polyphosphate4-phosphatase type II), a kind of lipid phosphatase. A potential tumor suppressor has abnormal expression of INPP4B in many malignant tumors, such as in leukemia, breast cancer, prostate cancer, melanoma, liver cancer, gastric cancer and other malignant tumors, it is found that the deletion of INPP4B or the abnormal expression of.INPP4B mainly dephosphorylation of the D4 position group of phosphatidylinositol 3,4- two phosphoric acid (PI (3,4) P2). The product is 3- phosphatidyl inositol (PI (3) P), and PI (3,4) P2 activates the AKT. When PI (3,4) P2 decreases, the role of the PI3K/AKT signal transduction pathway is suppressed, and INPP4B acts to inhibit the occurrence of the tumor. EN, PTEN) is a protein.PTEN, which is located on the chromosome 10q23.31, encoding the double phospholipase activity of phospholipid and protein consisting of 403 amino acids. It is one of the most hot tumor suppressor genes in cancer research. It is heavy in cell cycle regulation, inducing cell apoptosis and cell migration and adhesion. The regulatory effect of.PTEN protein is mainly dephosphorylated by dephosphorylation to dephosphoric the D3 bit group of phosphatidylinositol 3,4,5- three phosphoric acid (PI (3,4,5) P3) to regulate the PI (3,4,5) P3 level in the cell and to regulate the PI3K/AKT pathway. This experiment is to detect the expression of INPP4B and PTEN in human gliomas and the correlation between the two. At the molecular level, a preliminary discussion on the pathogenesis of glioma is made. At the same time, we hope to provide a theoretical basis for the diagnosis and classification of glioma, prognosis and treatment. Methods: 60 specimens of glioma resection and pathological diagnosis of glioma in Affiliated Hospital of Chuanbei Medical College Department of neurosurgery from January 2015 to August 2016 were collected. 33 male patients, 27 female patients, grade I glioma 5 cases, grade II glioma 26 cases, grade III glioma 20 cases and grade IV glioma 9 cases. All the above glioma specimens were classified according to the classification of central nervous system tumor of WHO in 2007, all the specimens had complete medical records, and no radiotherapy was performed before the operation. In addition, 25 cases of normal brain tissue specimens were collected as control analysis (the specimens were mainly derived from the contusion necrotic brain tissue in the patients with high intracranial pressure, such as brain trauma). After all the specimens were cut down, the blood was removed by repeated rinsing of the PBS liquid at about 4 degrees C, and the tissues of the necrotic and electrocauteric inactivation were removed. The specimens were divided into two parts. One was placed in the polycondensation Formaldehyde Solution in 4% to be fixed for 1 to 2 days later. The other was placed in the liquid nitrogen and transferred to the laboratory. Then it was stored in the refrigerator at -80 C. The immunohistochemistry (IHC) and real-time fluorescence quantitative PCR (qRT-PCR) method were used to determine the INPP4B and P in normal brain tissue and glioma respectively. The expression of TEN and statistical software SPSS22.0 were used to analyze the experimental data. Results: 1 the results of immunohistochemistry showed that the expression of INPP4B in the normal brain tissue and the low grade glioma group and the high grade glioma group were statistically significant (P0.05), and the positive rate was 92% in the normal brain tissue and the low grade glia. In the tumor group, 67.7% and 37.9% in the high grade glioma group, the expression of INPP4B in the low grade glioma group was higher than that of the high grade glioma group (P0.05). The expression of INPP4B in the normal brain tissue was higher than the low grade glioma group. The high grade glioma group (P0.05).PTEN was expressed in the normal brain tissue and the low grade glioma group, and the high grade glioma group was expressed in three groups. The difference was statistically significant (P0.05), and the positive rate was 96% in the normal brain tissue group, 74.2% in the low grade glioma group and 41.4% in the high grade glioma group; the PTEN expression in the low grade glioma group was higher than the high grade glioma group (P0.05), and the PTEN in the normal brain tissue was higher than the low grade glioma group, and the high grade glioma group (P0.05).2 qRT- The results of PCR experiment showed that the expression of INPP4B mRNA in the normal brain tissue and low grade glioma group, the expression of three groups in the high grade glioma group was statistically significant (P0.05). The expression level of INPP4B mRNA in the low grade glioma group was lower than that of the normal brain tissue group (P0.05). The expression of INPP4B mRNA in the high grade glioma group was lower than that of the lower grade gel group. The expression of PTEN mRNA in the normal brain tissue and the low grade glioma group and the high grade glioma group was statistically significant (P0.05). The expression level of PTEN mRNA in the low grade glioma group was lower than that of the normal brain group (P0.05), and the expression level of PTEN mRNA in the high grade glioma group was lower than that in the low grade glioma group, and the expression level of PTEN mRNA in the high grade glioma group was lower than that in the low grade glioma group. The expression level of PTEN mRNA in the high grade glioma group was lower than that in the low grade glioma group. Grade glioma group and normal brain tissue group (P0.05); INPP4B and PTEN were positively correlated in three groups of normal brain tissue, low grade glioma, and high grade glioma (r=0.752, P=0.000). Conclusion: the expression level of 1 INPP4B and PTEN is negatively correlated with the malignant grade of glioma, and the expression level of two of glioma is increased with the malignant degree of glioma. It is suggested that the two may have a positive correlation with the expression level of.2 INPP4B and PTEN in glioma. It is presumed that it may have synergistic effects in inhibiting the occurrence, development, invasion and proliferation of glioma,.3 based on the same downward trend in the expression of INPP4B and PTEN in glioma, so the association is inferred. Detection of INPP4B and PTEN may serve as a reference index for grading diagnosis and prognosis of glioma.

【学位授予单位】：川北医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41

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