骨膜蛋白促进恶性脑膜瘤侵袭与增殖的相关机制研究

发布时间：2018-04-24 02:01

本文选题：脑膜瘤 + 骨膜蛋白　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景与目的脑膜瘤是起源于蛛网膜帽状细胞的颅内最常见的非神经上皮性肿瘤。绝大多数脑膜瘤为良性肿瘤(WHO I级),但仍然有高达25%左右的脑膜瘤为非典型脑膜瘤(WHO II级)和恶性脑膜瘤(WHO III级)。恶性脑膜瘤的预后最差,中位生存期仅为1.5年,总体复发率高达80%,肿瘤的病理分级和侵袭是影响复发和预后的关键因素。因此,寻找新的有效治疗方法成为了目前改善恶性脑膜瘤预后的首要目标。在我们的前期研究中,发现骨膜蛋白(periostin,POSTN)在不同级别中的脑膜瘤间质中呈不同程度的表达,并且其表达量也随着病理等级的增高而增高。骨膜蛋白作为一种细胞外基质(extracellular matrix,ECM)蛋白,可与整合素受体相结合,参与肿瘤微环境的调控,促进肿瘤的侵袭与增殖,导致肿瘤发生上皮间质转化(epithelial-mesenchymal transition,EMT)等恶性行为。但骨膜蛋白在脑膜瘤中的作用及分子机制尚未有研究报道。本项研究通过对骨膜蛋白在恶性脑膜瘤中的分子机制的研究,探讨了促进其侵袭和增殖的信号通路及关键蛋白,可为恶性脑膜瘤的综合治疗提供潜在的治疗靶点及新的启示。研究内容与方法1.恶性脑膜瘤原代细胞的培养与鉴定收集新鲜标本进行原代细胞培养并纯化,观察原代细胞形态,测定增殖能力,免疫荧光鉴定细胞。2.骨膜蛋白对恶性脑膜瘤细胞功能及表型的研究不同级别的脑膜瘤组织进行qRT-PCR和Western-blot检测POSTN的表达量,免疫组化法观察POSTN在不同级别脑膜瘤中的表达程度及在恶性脑膜瘤中的表达部位,Spearman相关性检测法检测POSTN与恶行脑膜瘤侵袭的相关性,Western-blot检测分离纯化后的恶性脑膜瘤细胞与肿瘤相关成纤维细胞中POSTN的表达量。3.骨膜蛋白影响恶性脑膜瘤恶性表型的机制研究原代培养的恶性脑膜瘤细胞分别转染过表达POSTN病毒与空载病毒,Western-blot检测两组细胞EMT相关蛋白的变化情况以及Akt、pAkt的变化情况。研究结果1.原代培养的脑膜瘤细胞经鉴定EMA及Vimentin表达阳性,恶性脑膜瘤原代细胞增殖能力强于良性脑膜瘤细胞,良性脑膜瘤细胞中β-半乳糖苷酶染色阳性率更高。2.恶性脑膜瘤中POSTN蛋白水平和mRNA水平表达都高于良性脑膜瘤(P0.01),POSTN与恶性脑膜瘤侵袭显著相关(r=0.738,p0.001),POSTN表达于恶性脑膜瘤的间质中,原代培养中的成纤维细胞POSTN含量高于脑膜瘤细胞(P0.01)。3.恶性脑膜瘤细胞过表达POSTN后E-ca明显下调,N-ca、p-Akt、Vimentin、Snail明显上调(P0.001);增殖能力及侵袭能力增强(P0.001)。研究结论恶性脑膜瘤与良性脑膜瘤相比,具备更强的增殖能力,也更具有侵袭性,这可能是导致高级别脑膜瘤预后更差的原因。由高级别脑膜瘤中的肿瘤相关成纤维细胞分泌的细胞外基质蛋白——骨膜蛋白,可能是调控高级别脑膜瘤恶性表型的一个关键蛋白。骨膜蛋白可促进Akt的磷酸化使其相关通路激活,影响高级别脑膜瘤的EMT过程从而促进肿瘤的侵袭。因此,针对这一调控机制的靶向治疗,或许可以对高级别脑膜瘤的综合治疗提供新的靶点,改善恶性脑膜瘤患者的预后。
[Abstract]:Background and objective meningiomas are the most common non neuroepithelial tumors of the intracranial arachnoid cap cells. Most meningiomas are benign tumors (WHO I grade), but there are still about 25% of meningiomas as atypical meningiomas (WHO II) and malignant meningiomas (WHO III). The worst prognosis of malignant meningiomas is the middle stage of malignant meningioma. The survival time is only 1.5 years and the overall recurrence rate is up to 80%. The pathological classification and invasion of the tumor are the key factors affecting the recurrence and prognosis. Therefore, finding new effective treatments has become the primary goal of improving the prognosis of malignant meningioma. In our previous study, we found the meninges of Periostin (POSTN) in different levels. The expression of the tumor is expressed in varying degrees and its expression increases with the increase of the pathological grade. As an extracellular matrix (extracellular matrix, ECM) protein, periosteum protein can be combined with integrin receptor, participate in the regulation of tumor microenvironment, promote tumor invasion and proliferation, and lead to epithelial mesenchymal transformation of tumor (EP Ithelial-mesenchymal transition, EMT) and other malignant behaviors. However, the role and molecular mechanism of periosteum protein in meningioma have not been reported. This study has explored the signal pathways and key proteins that promote the invasion and proliferation of the periosteum protein in the malignant meningioma, and can be used as a synthesis of the malignant meningioma. Combination therapy provides potential therapeutic targets and new implications. Research contents and methods 1. primary cells of malignant meningioma are cultured and identified to collect fresh specimens for primary cell culture and purification, observe the morphology of primary cells, determine the proliferation ability, and immunofluorescence identification of.2. periosteum for the function and phenotype of malignant meningioma cells. The expression of POSTN was detected by qRT-PCR and Western-blot in different meningioma tissues. The expression level of POSTN in different meningiomas and the expression of POSTN in different meningiomas were observed by immunohistochemistry. The correlation between POSTN and the invasion of malignant meningioma was detected by Spearman correlation detection. Western-blot was used to detect the separation and purity of the meningioma. The expression of POSTN in malignant meningioma cells and tumor related fibroblasts.3. effect of periosteum protein on malignant phenotype of malignant meningioma; the primary cultured malignant meningioma cells were transfected with POSTN virus and airborne virus respectively. The changes of EMT related proteins in two groups of cells were detected by Western-blot and Akt, pA KT changes. Results 1. primary cultured meningioma cells were identified as positive for EMA and Vimentin. The primary cell proliferation of malignant meningioma was stronger than that of benign meningioma cells. The positive rate of beta galactosidase staining in benign meningioma cells was higher than that in.2. malignant meningioma. The expression of POSTN protein and mRNA was higher than that of benign meningioma. P0.01, POSTN was significantly associated with malignant meningioma (r=0.738, p0.001). POSTN was expressed in the stroma of malignant meningioma. The content of POSTN in primary cultured fibroblasts was higher than that of meningioma cells (P0.01).3. malignant meningioma cells. Colonization and invasiveness enhanced (P0.001). Conclusions malignant meningiomas are more proliferative and aggressive than benign meningiomas, which may be the cause of worse prognosis in the high grade meningioma. The extracellular matrix protein secreted by the tumor related fibroblast in the high level meningioma, the periosteum egg White, may be a key protein in the regulation of the malignant phenotype of high grade meningiomas. Periosteum can promote the phosphorylation of Akt to activate its pathway, affect the EMT process of the high level meningioma and promote the invasion of the tumor. Therefore, targeted therapy for this regulatory mechanism, or permits for the comprehensive treatment of high level meningiomas, may be provided. The new target is to improve the prognosis of patients with malignant meningioma.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.45

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