發(fā)布時(shí)間：2018-03-19 08:39

  本文選題：腦膠質(zhì)瘤　切入點(diǎn)：CMG2　出處：《第三軍醫(yī)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景和目的膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)常見(jiàn)的惡性腫瘤。盡管近年來(lái)其治療方法有了長(zhǎng)足的進(jìn)步,但其療效仍不盡人意。高度侵襲性是導(dǎo)致病人預(yù)后差的重要原因之一。因此,深入研究膠質(zhì)瘤侵襲的分子機(jī)制,對(duì)于發(fā)展有效的膠質(zhì)瘤治療新策略具有重要意義。毛細(xì)血管形態(tài)發(fā)生基因(Capillary morphogenesis gene 2,cmg2)因其在血管形成過(guò)程顯著上調(diào)表達(dá)而被發(fā)現(xiàn),因其編碼的蛋白能介導(dǎo)炭疽桿菌毒素進(jìn)入細(xì)胞內(nèi),故又稱(chēng)其為炭疽毒素受體2(Anthrax Toxin Receptor 2,ANTXR2),并被證明是炭疽桿菌毒素親和力最強(qiáng)的受體。業(yè)已闡明CMG2突變會(huì)導(dǎo)致幼年透明性纖維瘤病(Juvenile Hyaline Fibromatosis)。但CMG2確切的生理功能尚不十分清楚。近年來(lái),已有關(guān)于CMG2與腫瘤關(guān)系的報(bào)道,CMG2高表達(dá)能促進(jìn)前列腺癌的轉(zhuǎn)移。正常腦組織中不表達(dá)CMG2,至今也無(wú)CMG2與膠質(zhì)瘤關(guān)系的研究報(bào)道,但本課題組前期研究發(fā)現(xiàn)CMG2在膠質(zhì)瘤中顯著高表達(dá),并與腫瘤級(jí)別呈正相關(guān)而與病人預(yù)后呈負(fù)相關(guān)。鑒于侵襲性是膠質(zhì)瘤最顯著的惡性生物學(xué)行為之一,本課題研究的目的在于探索CMG2在膠質(zhì)瘤侵襲中的作用及可能的的分子機(jī)制,從而有望加深對(duì)膠質(zhì)瘤侵襲機(jī)制的認(rèn)識(shí),也可能為膠質(zhì)瘤的預(yù)后判斷提供新的指標(biāo)并為臨床治療提供新的靶點(diǎn)。主要方法1.利用慢病毒方法,構(gòu)建穩(wěn)定過(guò)表達(dá)CMG2的膠質(zhì)瘤細(xì)胞U87 OE和091214 OE以及穩(wěn)定敲低CMG2表達(dá)的膠質(zhì)瘤細(xì)胞U87-sh2。2.Matrigel-transwell侵襲實(shí)驗(yàn)觀察過(guò)表達(dá)/敲低CMG2細(xì)胞侵襲能力的差異,劃痕實(shí)驗(yàn)觀察其遷移能力的改變。3.建立小鼠原位移植瘤模型,觀察過(guò)表達(dá)CMG2與否的膠質(zhì)瘤細(xì)胞在體內(nèi)的侵襲能力的差異以及對(duì)小鼠生存期的影響。4.細(xì)胞免疫熒光方法用于觀察不同CMG2表達(dá)狀態(tài)膠質(zhì)瘤細(xì)胞絲狀偽足和層狀偽足的差異。5.Western Blot檢測(cè)比較CMG2過(guò)表達(dá)與否的膠質(zhì)瘤細(xì)胞中YAP及p-YAP的表達(dá)水平。6.免疫組織化學(xué)方法分析72例不同級(jí)別的膠質(zhì)瘤病人腫瘤組織中YAP的表達(dá)特征,采用Kaplan-Meier法和LogRank檢驗(yàn)分析YAP在膠質(zhì)瘤組織中的表達(dá)與病人預(yù)后的關(guān)系。結(jié)果1.CMG2促進(jìn)膠質(zhì)瘤細(xì)胞侵襲和遷移能力(1)體外Matrigel-transwell侵襲實(shí)驗(yàn)和劃痕實(shí)驗(yàn)顯示:與對(duì)照組相比較,過(guò)表達(dá)CMG2增強(qiáng)膠質(zhì)瘤細(xì)胞侵襲和遷移能力(p0.05),而敲低CMG2則降低膠質(zhì)瘤細(xì)胞的遷移能力(p0.05)。(2)小鼠體內(nèi)原位移植瘤實(shí)驗(yàn)顯示:與對(duì)照組相比較,種植了過(guò)表達(dá)CMG2的膠質(zhì)瘤細(xì)胞的小鼠腫瘤組織前沿具明顯侵襲現(xiàn)象,小鼠生存期縮短(p0.05);種植CMG2下調(diào)表達(dá)細(xì)胞的小鼠生存期延長(zhǎng)(p0.05)。2.CMG2促進(jìn)膠質(zhì)瘤細(xì)胞層狀偽足和絲狀偽足的形成細(xì)胞免疫熒光染色分析發(fā)現(xiàn):與對(duì)照組相比較,過(guò)表達(dá)CMG2的膠質(zhì)瘤細(xì)胞能形成更多的層狀偽足和絲狀偽足。3.CMG2上調(diào)膠質(zhì)瘤細(xì)胞中YAP的表達(dá)Western Blot檢測(cè)分析發(fā)現(xiàn):伴隨過(guò)表達(dá)CMG2的膠質(zhì)瘤細(xì)胞偽足形成增多,Hippo通路的關(guān)鍵效應(yīng)蛋白YAP的表達(dá)上調(diào)。4.膠質(zhì)瘤組織中YAP的表達(dá)與腫瘤級(jí)別呈正相關(guān)而與患者預(yù)后呈負(fù)相關(guān)(1)免疫組織化學(xué)分析顯示:YAP在高級(jí)別膠質(zhì)瘤中的表達(dá)高于低級(jí)別(p0.05)。(2)生存分析顯示:YAP高表達(dá)的膠質(zhì)瘤病人預(yù)后不良(p0.05)。結(jié)論1.CMG2對(duì)膠質(zhì)瘤細(xì)胞侵襲能力具有促進(jìn)作用。2.CMG2可能通過(guò)上調(diào)YAP表達(dá)誘導(dǎo)偽足形成進(jìn)而促進(jìn)膠質(zhì)瘤細(xì)胞侵襲。3.人膠質(zhì)瘤組織中YAP的表達(dá)與腫瘤級(jí)別呈正相關(guān),與膠質(zhì)瘤病人預(yù)后呈負(fù)相關(guān)。
[Abstract]:Background and objective: glioma is the most common malignant tumors of the central nervous system. In recent years although the treatment method has made great progress, but its effect is still unsatisfactory. Highly invasive is an important cause of poor prognosis of patients. Therefore, further study of molecular mechanism of glioma invasion, is of great significance for the new strategy for the treatment of glioma the development of tumor effectively. Capillary morphogenesis gene (Capillary morphogenesis gene 2, cmg2) due to the upregulation of vascular formation was found, because of its encoding protein can mediate the anthrax toxin into cells, it is also known as anthrax toxin receptor 2 (Anthrax Toxin 2 Receptor, ANTXR2), and anthrax toxin was proved to be the highest affinity receptor. It is already illustrated CMG2 mutations can lead to juvenile hyaline fibromatosis (Juvenile Hyaline Fibromatosis). But the exact physiological CMG2 Function is not very clear. In recent years, has been reported about the relationship between CMG2 and tumor, the high expression of CMG2 can promote the metastasis of prostate cancer. The expression of CMG2 in normal brain tissue, the research report has no relationship between CMG2 and glioma, but the previous study found that CMG2 in glioma tissues, and was positively correlated with tumor grade and negatively correlated with the prognosis of the patients. In view of the invasive biological behavior of malignant glioma is one of the most significant, the purpose of this study is to explore the effect of CMG2 on the invasion of glioma cells and the possible molecular mechanism, which is expected to deepen the understanding on the mechanism of the invasion of glioma, may provide new indicators and provide a new target for clinical treatment for glioma prognosis judgment. The 1. main methods of using lentiviral method, construct the stable over expression of CMG2 glioma cells U87 OE and 091214 OE and stability On U87-sh2.2.Matrigel-transwell glioma cells with low CMG2 expression of invasion experiment observed differential expression / invasive ability of CMG2 cells at low, scratch test to observe the migration ability change.3. mice orthotopic transplantation tumor model, observe the expression of CMG2 and glioma cells in vivo invasion ability difference and the survival of the mice.4. immunofluorescence methods were used to analyze the expression features of 72 cases with different levels of glioma patients in tumor tissue YAP expression level of.6. immunohistochemical method between.5.Western Blot detection to observe the different expression of CMG2 in glioma cells and filopodia between CMG2 YAP lamellar pseudopodia and the expression of p-YAP and glioma cells, the relationship between by using Kaplan-Meier method and LogRank test to analyze the expression of YAP and prognosis in glioma tissues. The results showed that 1.CMG2 promotes glioma Tumor cell invasion and migration (1) showed that in vitro Matrigel-transwell assay and scratch test: compared with the control group, overexpression of CMG2 enhances the invasion and migration of glioma cells (P0.05), while knockdown of CMG2 decreased the migration ability of glioma cells (P0.05). (2) showed in mice results: compared with the control group, planting overexpression glioma cells CMG2 mice tumor tissue invasion frontier has obvious phenomenon, shorten the survival time of mice (P0.05); cultivation of down regulated expression of CMG2 cells of mice prolonged survival (P0.05).2.CMG2 promotes glioma cells and filopodia formation of lamellar pseudopodia cell immunofluorescence staining the analysis found that: compared with the control group, expression of Western Blot detected CMG2 glioma cells can form more lamellar pseudopodia and filopodia upregulation of.3.CMG2 glioma cells in YAP analysis Now with over expression of glioma cell pseudopod formation of CMG2 was increased, and the expression of tumor associated protein YAP level key effect of Hippo pathway up-regulated the expression of.4. in human glioma YAP and prognosis in patients with negative correlation (1) immunohistochemical analysis showed that the expression of YAP in high grade gliomas was higher than that of low level (P0.05). (2) survival analysis showed that the prognosis of patients with glioma YAP high expression of bad (P0.05). Conclusion 1.CMG2 can promote the.2.CMG2 may regulate YAP expression and tumor grade was positively induced podosome formation and promote invasion of.3. human glioma tissues YAP glioma cells related to glioma cell invasion ability, and negatively related to the prognosis of glioma patients.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R739.41

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相關(guān)期刊論文 前1條

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本文編號(hào)：1633498