本文選題：膠質(zhì)瘤 + miR-451��； 參考：《天津醫(yī)科大學》2014年碩士論文

【摘要】：膠質(zhì)母細胞瘤(glioblastoma, GBM)是侵襲性最強的惡性膠質(zhì)瘤,也是最常見的原發(fā)性中樞神經(jīng)系統(tǒng)腫瘤,屬人類預后極差的腫瘤之一。如果僅進行支持治療,膠質(zhì)母細胞瘤病人的中位生存期不足3個月,97%的病人在12月內(nèi)死亡；對于手術(shù)治療病人,由于腫瘤細胞在腦實質(zhì)內(nèi)呈彌漫浸潤性生長,常不能實現(xiàn)滿意的擴大切除。面對并不樂觀的現(xiàn)狀,現(xiàn)代醫(yī)學工作者依然不懈的致力于對膠質(zhì)母細胞瘤治療手段的研究。近十余年來,微創(chuàng)理念和影像導引外科等新技術(shù)的不斷發(fā)展,顯著提高了惡性腦腫瘤影像學全切除率,并降低了術(shù)后致殘、致死率,同時各種改良的綜合治療措施和新療法也為膠質(zhì)母細胞瘤的治療帶來了新希望。目前,盡管腫瘤切除手術(shù)后替莫唑胺(temozolomide,TMZ)聯(lián)合常規(guī)分割照射的標準治療方案有了值得關(guān)注的發(fā)展,接受標準方案治療的新診斷的膠質(zhì)母細胞瘤患者相對于單純放療的患者二年生存率由10.9%提高到27.2%,三年生存率由4.4%提高到16%,但膠質(zhì)母細胞瘤的侵襲遷移生物學特性依然嚴重制約著各種治療手段的進一步提高。 腫瘤細胞的增殖和運動密切相關(guān)。早在1996年就有研究提出了腫瘤細胞的"go or grow'假說。當腫瘤細胞所處的局部微生態(tài)環(huán)境不利于腫瘤細胞的增殖時,腫瘤細胞會遷移至適宜腫瘤細胞生存和增殖的環(huán)境。在這一過程中,腫瘤細胞在適合的微環(huán)境中進行增殖與遷移表型的轉(zhuǎn)換,啟動相應的生物學變化。盡管對腫瘤細胞遷移運動和增殖轉(zhuǎn)換的分子調(diào)控機制還很不清楚,但腫瘤細胞的運動與增殖表型轉(zhuǎn)換肯定不是受外部因素單獨影響的,在細胞增殖和細胞運動兩種表型復雜的分子和信號通路調(diào)控之間,細胞內(nèi)應該存在一個表觀遺傳學的調(diào)控開關(guān)。微小RNA (microRNA, miRNA, miR)是一種內(nèi)源性的非編碼RNA,對基因表達進行抑制性調(diào)控,調(diào)節(jié)包括增殖,分化,凋亡等多種腫瘤細胞的發(fā)生、發(fā)展過程。最近研究表明,miR-451在遷移運動的膠質(zhì)瘤細胞中明顯下調(diào),miR-451調(diào)節(jié)LKB1-AMPK通路,可能使膠質(zhì)瘤細胞在不同的葡萄糖環(huán)境中分別表現(xiàn)出增殖活性和遷移運動活性。 本研究主要討論miR-451對膠質(zhì)瘤細胞增殖和運動的作用,對膠質(zhì)瘤細胞增殖相關(guān)蛋白mTOR和遷移調(diào)控蛋白Rac1活性的影響,以及miR-451調(diào)控AMPK, mTOR, Racl蛋白的激活狀態(tài)的潛在機制。 本課題研究分為以下兩個部分： 第一部分明確miR-451在人腦膠質(zhì)瘤中的表達情況,觀察miRNA-451對膠質(zhì)瘤細胞增殖和運動的影響。我們收集40例膠質(zhì)母細胞瘤(WHO IV)標本以及6例顳葉癲癇手術(shù)患者的對照腦組織。本課題組運用qRT-PCR檢測不同標本中miR-451的表達情況。通過將miR-451類似物和抑制物轉(zhuǎn)染U87、U251、SNB19三個人腦膠質(zhì)瘤細胞系,探討miRNA-451對膠質(zhì)瘤細胞增殖和運動能力的作用。qRT-PCR驗證轉(zhuǎn)染效果；MTT實驗檢測細胞增殖活性；體外劃痕實驗和transwell遷移實驗檢測細胞的遷移能力。結(jié)果顯示：膠質(zhì)母細胞瘤組織中miR-451的相對表達量是對照腦組織的(33.58±5.19)%；MTT實驗表明miR-451對膠質(zhì)瘤細胞增殖能力呈正性調(diào)節(jié)作用；劃痕實驗和transwell遷移實驗發(fā)現(xiàn)miR-451對膠質(zhì)瘤細胞運動能力呈負性調(diào)節(jié)作用。 第二部分miR-451對膠質(zhì)瘤細胞增殖及運動能力作用的潛在機制。1.課題組將miR-451類似物和抑制物轉(zhuǎn)染進入U87、U251、SNB19三個人腦膠質(zhì)瘤細胞系,運用qRT-PCR驗證轉(zhuǎn)染效果；western blot技術(shù)檢測不同處理組細胞中p-AMPK、AMPK、p-Raptor、Raptor、Racl的表達；GST-pulldown技術(shù)檢測GTP-Racl表達。結(jié)果顯示：各組細胞中AMPK、Raptor、Racl的表達與miR-451的表達無明確相關(guān)性;p-AMPK、GTP-Racl的表達量與miR-451表達呈負相關(guān),與p-Raptor的表達呈正相關(guān)。2.為了進一步驗證結(jié)論,我們通過RNA干擾技術(shù)沉默U87、U251、SNB19三個人腦膠質(zhì)瘤細胞系中AMPKal的表達,在此基礎(chǔ)上轉(zhuǎn)染miR-451類似物和抑制物,qRT-PCR和western blot驗證轉(zhuǎn)染效果；MTT實驗檢測細胞增殖活性；體外劃痕實驗和transwell遷移實驗檢測細胞的遷移能力；western blot技術(shù)檢測不同處理組中p-AMPK、AMPK、p-Raptor、Raptor、Rac1的表達；GST-pulldown技術(shù)檢測GTP-Racl表達。結(jié)果顯示：敲低AMPKal后,miR-451對細胞增殖,遷移能力及相關(guān)蛋白p-Raptor、GTP-Racl的影響不同程度減小甚至消失。 結(jié)論： 1.miR-451在膠質(zhì)母細胞瘤腫瘤組織標本的miR-451的表達量顯著低于對照腦組織標本。體外實驗證實,miR-451增強膠質(zhì)瘤細胞的增殖能力,同時抑制膠質(zhì)瘤細胞的遷移能力。 2.miR-451通過調(diào)節(jié)AMPK的激活,調(diào)控Rac1和mTORC1活性,從而影響膠質(zhì)瘤細胞的增殖能力和遷移運動能力。
[Abstract]:Glioblastoma (GBM) is the most invasive glioma. It is also one of the most common primary central nervous system tumors. It is one of the most malignant tumors in human prognosis. If only support treatment, the median survival time of the patients with glioblastoma is less than 3 months, and 97% of the patients died in December. Patients, due to the diffuse infiltrating growth of tumor cells in the brain parenchyma, are often unable to achieve satisfactory expanded excision. Facing the unoptimistic situation, modern medical workers are still unremitting efforts to study the treatment of glioblastoma. In the past ten years, new techniques such as minimally invasive ideas and imaging guidance surgery have developed continuously. The total resection rate of malignant brain tumors is improved, and the postoperative disability and mortality rate are reduced. Meanwhile, various improved comprehensive treatments and new treatments have also brought new hope for the treatment of glioblastoma. Currently, the standard treatment regimen of temozolomide, TMZ combined with conventional fractionated irradiation after tumor resection is available. The two year survival rate of the newly diagnosed glioblastoma patients with the standard regimen treatment increased from 10.9% to 27.2%, and the three year survival rate increased from 4.4% to 16%, but the biological characteristics of the invasion and migration of glioblastoma still severely restricted the further improvement of various treatments.
The proliferation and movement of tumor cells are closely related. A "go or GROW'hypothesis" was proposed in 1996. When the local microecological environment in the tumor cells is not conducive to the proliferation of tumor cells, the tumor cells migrate to the environment suitable for the survival and proliferation of tumor cells. In this process, the tumor cells are suitable for the tumor cells. In the microenvironment, the transformation of proliferation and migration phenotypes is carried out, and the corresponding biological changes are initiated. Although the molecular regulation mechanism of the migration and proliferation of tumor cells is not clear, the transformation of the tumor cell's movement and proliferation phenotype must not be affected by external factors alone, in cell proliferation and cell movement two phenotypes. Between complex molecular and signal transduction pathways, there should be a regulatory switch in epigenetics. Micro RNA (microRNA, miRNA, miR) is an endogenous non coded RNA that regulates the expression of genes and regulates the occurrence and development of a variety of tumor cells including proliferation, differentiation and apoptosis. Recent studies have shown that MiR-451 is obviously down regulated in the glioma cells of the migratory movement, and miR-451 regulates the LKB1-AMPK pathway, which may show the proliferation and mobility activity of glioma cells in different glucose environments.
The purpose of this study is to discuss the effect of miR-451 on the proliferation and movement of glioma cells, the effect of the proliferation related protein mTOR on glioma cells and the activity of migration regulatory protein Rac1, and the potential mechanism of miR-451 regulation of the activation state of AMPK, mTOR, and Racl proteins.
This research is divided into the following two parts:
The first part is to clarify the expression of miR-451 in human glioma and observe the effect of miRNA-451 on the proliferation and movement of glioma cells. We collect 40 cases of glioblastoma (WHO IV) and 6 cases of temporal lobe epilepsy surgery. The study group used qRT-PCR to detect the expression of miR-451 in different specimens. MiR-451 analogues and inhibitors were transfected into U87, U251, SNB19 three human glioma cell lines to explore the effect of miRNA-451 on the proliferation and movement of glioma cells by.QRT-PCR; MTT test was used to detect cell proliferation activity; in vitro scratch test and Transwell migration test to detect cell migration ability. The results showed: glue The relative expression of miR-451 in the tissue of the blastoma was (33.58 + 5.19)% of the control brain tissue. The MTT experiment showed that miR-451 had a positive regulating effect on the proliferation ability of glioma cells, and the scratch test and Transwell migration test showed that miR-451 had a negative regulating effect on the motor ability of glioma cells.
The potential mechanism of the second part miR-451 on the proliferation and motor ability of glioma cells.1. subjects transfected miR-451 analogues and inhibitors into U87, U251, SNB19 three human glioma cell lines, using qRT-PCR to verify the transfection effect; Western blot technique was used to detect p-AMPK, AMPK, p-Raptor, and expressions in different treatment groups. The expression of AMPK, Raptor, Racl in each group had no definite correlation with the expression of miR-451; the expression of p-AMPK, GTP-Racl was negatively correlated with the expression of miR-451, and was positively correlated with the expression of p-Raptor, and was positively correlated with the expression of p-Raptor. In order to further verify the conclusion, we were silent by RNA interference. The expression of AMPKal in SNB19 three human glioma cell lines, on this basis, transfection of miR-451 analogue and inhibitor, qRT-PCR and Western blot to verify the transfection effect; MTT test was used to detect cell proliferation activity; in vitro scratch test and Transwell migration test to detect cell migration energy; Western blot technique was used to detect p- in different treatment groups. The expression of AMPK, AMPK, p-Raptor, Raptor, Rac1; GST-pulldown technology to detect GTP-Racl expression. The results showed that after knocking low AMPKal, the effect of miR-451 on cell proliferation, mobility and associated protein p-Raptor, GTP-Racl was reduced and even disappeared in varying degrees.
Conclusion:
The expression of 1.miR-451 in the miR-451 of glioblastoma tumor tissue was significantly lower than that of the control brain tissue. In vitro experiments confirmed that miR-451 enhanced the proliferation ability of glioma cells and inhibited the migration ability of glioma cells.
2.miR-451 regulates the activity of Rac1 and mTORC1 by regulating the activation of AMPK, thereby affecting the proliferation and migration ability of glioma cells.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.41

【引證文獻】

相關(guān)碩士學位論文 前1條

1 高志奎;MiR-144-451基因簇與食管癌發(fā)病關(guān)系研究[D];東南大學;2016年

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本文編號：1822155