本文選題：膠質(zhì)瘤 + INPP4B�。� 參考：《川北醫(yī)學院》2017年碩士論文

【摘要】：背景及目的:人腦膠質(zhì)瘤多起源于神經(jīng)膠質(zhì)細胞,屬于神經(jīng)上皮源性腫瘤,約占中樞神經(jīng)系統(tǒng)中所有腫瘤的27%;在顱內(nèi)原發(fā)性惡性腫瘤中膠質(zhì)瘤是最常見的,約占80%。膠質(zhì)母細胞瘤(Glioblastoma,GBM,WHOⅣ級)在整個中樞神經(jīng)系統(tǒng)原發(fā)性惡性腫瘤中,發(fā)病率是最高的,占到46%左右,男性發(fā)病率高于女性。膠質(zhì)瘤具有起病隱匿,生長迅速,惡性程度相對較高,無明顯邊界,且呈浸潤性生長等特點,目前治療效果總體欠佳,在顱內(nèi)原發(fā)性腫瘤中,膠質(zhì)瘤患者的病死率是最高的,僅有1年左右的中位生存時間。與所有惡性腫瘤一樣,膠質(zhì)瘤的發(fā)生發(fā)展是一個多步驟、多因素的過程,但目前確切的發(fā)生機制仍有待進一步研究;伴隨著科學技術(shù)的進步,與膠質(zhì)瘤發(fā)生發(fā)展密切相關(guān)的腫瘤抑制因子越來越多的被發(fā)現(xiàn),這對于尋找針對膠質(zhì)瘤的靶基因或靶蛋白,以及膠質(zhì)瘤診斷分級分型、開發(fā)分子靶向藥物和判斷預(yù)后等具有極大的推動作用。在目前的研究基礎(chǔ)上,已發(fā)現(xiàn)以下3條基因信號通路的異�？赡苁悄z質(zhì)瘤發(fā)生發(fā)展的部分因素,包括:EGFR/PTEN/PI3K途徑,Rb-E2F/CDK4、6/P16-cyclin D途徑,P53/MDM2/P12、P21途徑。INPP4B基因是定位于人染色體4q31.21的一個潛在腫瘤抑制基因,其編碼的產(chǎn)物為磷脂酰肌醇4-磷酸酶Ⅱ型(INPP4B,inositol polyphosphate4-phosphatase type II),是一種脂質(zhì)磷酸酶。根據(jù)已有的研究結(jié)果顯示,作為一個潛在的腫瘤抑制因子,在許多惡性腫瘤中存在INPP4B的異常表達,如在白血病、乳腺癌、前列腺癌、黑色素瘤、肝癌、胃癌等惡性腫瘤中均發(fā)現(xiàn)INPP4B的表達缺失或表達異常。INPP4B主要使磷脂酰肌醇3,4-二磷酸(PI(3,4)P2)的D4位基團去磷酸化,其產(chǎn)物為3-磷酸磷脂酰肌醇(PI(3)P);而PI(3,4)P2能使AKT激活,當PI(3,4)P2降低時,則PI3K/AKT信號傳導通路作用被抑制,從而INPP4B起到抑制腫瘤的發(fā)生。人第10號染色體缺失的磷酸酶和張力蛋白同源物基因(phosphatase and tensin homolog deleted on chromosome ten,PTEN)是一個定位于染色體10q23.31,編碼由403個氨基酸組成的具有磷脂和蛋白質(zhì)的雙重磷脂酶活性的蛋白。PTEN作為目前在腫瘤研究中較為火熱的抑癌基因之一,其在細胞周期的調(diào)控、誘導細胞的凋亡以及細胞的遷移粘附等方面具有重要的調(diào)控作用。PTEN蛋白主要通過去磷酸化作用,使磷脂酰肌醇3,4,5-三磷酸(PI(3,4,5)P3)的D3位基團脫磷酸,來調(diào)節(jié)細胞內(nèi)PI(3,4,5)P3水平,對PI3K/AKT途徑進行負調(diào)控。本實驗擬通過檢測INPP4B與PTEN在人腦膠質(zhì)瘤中的表達情況及二者的相關(guān)性,以期能在分子水平對膠質(zhì)瘤發(fā)生發(fā)展機制作初步探討,同時希望能對膠質(zhì)瘤的診斷分級、預(yù)后判斷及治療等方面提供理論依據(jù)。方法:收集川北醫(yī)學院附屬醫(yī)院神經(jīng)外科2015年1月到2016年8月膠質(zhì)瘤手術(shù)切除并且病理明確診斷的標本60例,其中男性患者33例,女性患者27例,Ⅰ級膠質(zhì)瘤5例,Ⅱ級膠質(zhì)瘤26例,Ⅲ級膠質(zhì)瘤20例,Ⅳ級膠質(zhì)瘤9例。以上膠質(zhì)瘤標本均按照2007年WHO的中樞神經(jīng)系統(tǒng)腫瘤分類分級來進行病理分類分級,所收集標本均有完整病歷資料,且術(shù)前未進行放射治療、化學治療等抗腫瘤治療。另外再收集正常腦組織標本25例作為對照分析(標本主要來源于腦外傷等高顱內(nèi)壓患者手術(shù)時切除的挫傷壞死腦組織)。所有標本切下后,立即予以4℃左右PBS液反復沖洗去除血液,清除壞死、電灼失活的組織,然后將標本一分為二,一份置于4%的多聚甲醛溶液中固定1到2日后石蠟包埋備用,另一份放于液氮中轉(zhuǎn)移至實驗室,然后置于-80℃冰箱中保存?zhèn)溆�。采用免疫組織化學法(IHC)和實時熒光定量PCR(qRT-PCR)法分別測定正常腦組織和膠質(zhì)瘤中INPP4B和PTEN的表達情況;并采用統(tǒng)計軟件SPSS22.0對實驗數(shù)據(jù)進行統(tǒng)計分析。結(jié)果:1免疫組化實驗結(jié)果顯示:INPP4B在正常腦組織組和低級別膠質(zhì)瘤組、高級別膠質(zhì)瘤組三組間表達差異有統(tǒng)計學意義(P0.05);其陽性率:正常腦組織組中為92%,低級別膠質(zhì)瘤組中為67.7%,高級別膠質(zhì)瘤組中為37.9%;低級別膠質(zhì)瘤組中INPP4B表達高于高級別膠質(zhì)瘤組(P0.05),正常腦組織組中INPP4B表達高于低級別膠質(zhì)瘤組、高級別膠質(zhì)瘤組(P0.05)。PTEN在正常腦組織組和低級別膠質(zhì)瘤組、高級別膠質(zhì)瘤組三組間表達差異有統(tǒng)計學意義(P0.05);其陽性率:正常腦組織組中為96%,低級別膠質(zhì)瘤組中為74.2%,高級別膠質(zhì)瘤組中為41.4%;低級別膠質(zhì)瘤組中PTEN表達高于高級別膠質(zhì)瘤組(P0.05),正常腦組織中PTEN表達高于低級別膠質(zhì)瘤組、高級別膠質(zhì)瘤組(P0.05)。2 qRT-PCR實驗結(jié)果顯示:INPP4B mRNA在正常腦組織組和低級別膠質(zhì)瘤組、高級別膠質(zhì)瘤組三組間表達差異有統(tǒng)計學意義(P0.05),低級別膠質(zhì)瘤組中INPP4B mRNA的表達水平低于正常腦組織組(P0.05),高級別膠質(zhì)瘤組中INPP4B mRNA的表達水平低于低級別膠質(zhì)瘤組與正常腦組織組(P0.05);PTEN mRNA在正常腦組織組和低級別膠質(zhì)瘤組、高級別膠質(zhì)瘤組三組間表達差異有統(tǒng)計學意義(P0.05),低級別膠質(zhì)瘤組中PTEN mRNA的表達水平低于正常腦組織組(P0.05),高級別膠質(zhì)瘤組中PTEN mRNA的表達水平低于低級別膠質(zhì)瘤組與正常腦組織組(P0.05);INPP4B與PTEN在正常腦組織、低級別膠質(zhì)瘤、高級別膠質(zhì)瘤三組中表達成正相關(guān),(r=0.752,P=0.000)。結(jié)論:1 INPP4B與PTEN的表達水平均與膠質(zhì)瘤的惡性級別呈負相關(guān),隨著膠質(zhì)瘤惡性程度的增高二者的表達水平降低,提示二者可能為膠質(zhì)瘤的抑制基因。2 INPP4B與PTEN在膠質(zhì)瘤中的表達水平具有正相關(guān)性,推測其可能在抑制膠質(zhì)瘤的發(fā)生、發(fā)展、侵襲增殖等方面具有協(xié)同作用。3基于INPP4B與PTEN在膠質(zhì)瘤中的表達具有相同的下降趨勢,因此推斷聯(lián)合檢測INPP4B與PTEN可能作為膠質(zhì)瘤診斷分級、預(yù)后判斷等方面的參考指標。
[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.

【學位授予單位】：川北醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.41

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