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

【摘要】：膠質(zhì)母細(xì)胞瘤(glioblastoma, GBM)是侵襲性最強(qiáng)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(yù)后極差的腫瘤之一。如果僅進(jìn)行支持治療,膠質(zhì)母細(xì)胞瘤病人的中位生存期不足3個(gè)月,97%的病人在12月內(nèi)死亡；對(duì)于手術(shù)治療病人,由于腫瘤細(xì)胞在腦實(shí)質(zhì)內(nèi)呈彌漫浸潤性生長,常不能實(shí)現(xiàn)滿意的擴(kuò)大切除。面對(duì)并不樂觀的現(xiàn)狀,現(xiàn)代醫(yī)學(xué)工作者依然不懈的致力于對(duì)膠質(zhì)母細(xì)胞瘤治療手段的研究。近十余年來,微創(chuàng)理念和影像導(dǎo)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(xué)全切除率,并降低了術(shù)后致殘、致死率,同時(shí)各種改良的綜合治療措施和新療法也為膠質(zhì)母細(xì)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(biāo)準(zhǔn)治療方案有了值得關(guān)注的發(fā)展,接受標(biāo)準(zhǔn)方案治療的新診斷的膠質(zhì)母細(xì)胞瘤患者相對(duì)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(xì)胞瘤的侵襲遷移生物學(xué)特性依然嚴(yán)重制約著各種治療手段的進(jìn)一步提高。 腫瘤細(xì)胞的增殖和運(yùn)動(dòng)密切相關(guān)。早在1996年就有研究提出了腫瘤細(xì)胞的"go or grow'假說。當(dāng)腫瘤細(xì)胞所處的局部微生態(tài)環(huán)境不利于腫瘤細(xì)胞的增殖時(shí),腫瘤細(xì)胞會(huì)遷移至適宜腫瘤細(xì)胞生存和增殖的環(huán)境。在這一過程中,腫瘤細(xì)胞在適合的微環(huán)境中進(jìn)行增殖與遷移表型的轉(zhuǎn)換,啟動(dòng)相應(yīng)的生物學(xué)變化。盡管對(duì)腫瘤細(xì)胞遷移運(yùn)動(dòng)和增殖轉(zhuǎn)換的分子調(diào)控機(jī)制還很不清楚,但腫瘤細(xì)胞的運(yùn)動(dòng)與增殖表型轉(zhuǎn)換肯定不是受外部因素單獨(dú)影響的,在細(xì)胞增殖和細(xì)胞運(yùn)動(dòng)兩種表型復(fù)雜的分子和信號(hào)通路調(diào)控之間,細(xì)胞內(nèi)應(yīng)該存在一個(gè)表觀遺傳學(xué)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(duì)基因表達(dá)進(jìn)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(xì)胞的發(fā)生、發(fā)展過程。最近研究表明,miR-451在遷移運(yùn)動(dòng)的膠質(zhì)瘤細(xì)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(xì)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(yùn)動(dòng)活性。 本研究主要討論miR-451對(duì)膠質(zhì)瘤細(xì)胞增殖和運(yùn)動(dòng)的作用,對(duì)膠質(zhì)瘤細(xì)胞增殖相關(guān)蛋白mTOR和遷移調(diào)控蛋白R(shí)ac1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(jī)制。 本課題研究分為以下兩個(gè)部分： 第一部分明確miR-451在人腦膠質(zhì)瘤中的表達(dá)情況,觀察miRNA-451對(duì)膠質(zhì)瘤細(xì)胞增殖和運(yùn)動(dòng)的影響。我們收集40例膠質(zhì)母細(xì)胞瘤(WHO IV)標(biāo)本以及6例顳葉癲癇手術(shù)患者的對(duì)照腦組織。本課題組運(yùn)用qRT-PCR檢測不同標(biāo)本中miR-451的表達(dá)情況。通過將miR-451類似物和抑制物轉(zhuǎn)染U87、U251、SNB19三個(gè)人腦膠質(zhì)瘤細(xì)胞系,探討miRNA-451對(duì)膠質(zhì)瘤細(xì)胞增殖和運(yùn)動(dòng)能力的作用。qRT-PCR驗(yàn)證轉(zhuǎn)染效果；MTT實(shí)驗(yàn)檢測細(xì)胞增殖活性；體外劃痕實(shí)驗(yàn)和transwell遷移實(shí)驗(yàn)檢測細(xì)胞的遷移能力。結(jié)果顯示：膠質(zhì)母細(xì)胞瘤組織中miR-451的相對(duì)表達(dá)量是對(duì)照腦組織的(33.58±5.19)%；MTT實(shí)驗(yàn)表明miR-451對(duì)膠質(zhì)瘤細(xì)胞增殖能力呈正性調(diào)節(jié)作用；劃痕實(shí)驗(yàn)和transwell遷移實(shí)驗(yàn)發(fā)現(xiàn)miR-451對(duì)膠質(zhì)瘤細(xì)胞運(yùn)動(dòng)能力呈負(fù)性調(diào)節(jié)作用。 第二部分miR-451對(duì)膠質(zhì)瘤細(xì)胞增殖及運(yùn)動(dòng)能力作用的潛在機(jī)制。1.課題組將miR-451類似物和抑制物轉(zhuǎn)染進(jìn)入U(xiǎn)87、U251、SNB19三個(gè)人腦膠質(zhì)瘤細(xì)胞系,運(yùn)用qRT-PCR驗(yàn)證轉(zhuǎn)染效果；western blot技術(shù)檢測不同處理組細(xì)胞中p-AMPK、AMPK、p-Raptor、Raptor、Racl的表達(dá)；GST-pulldown技術(shù)檢測GTP-Racl表達(dá)。結(jié)果顯示：各組細(xì)胞中AMPK、Raptor、Racl的表達(dá)與miR-451的表達(dá)無明確相關(guān)性;p-AMPK、GTP-Racl的表達(dá)量與miR-451表達(dá)呈負(fù)相關(guān),與p-Raptor的表達(dá)呈正相關(guān)。2.為了進(jìn)一步驗(yàn)證結(jié)論,我們通過RNA干擾技術(shù)沉默U87、U251、SNB19三個(gè)人腦膠質(zhì)瘤細(xì)胞系中AMPKal的表達(dá),在此基礎(chǔ)上轉(zhuǎn)染miR-451類似物和抑制物,qRT-PCR和western blot驗(yàn)證轉(zhuǎn)染效果；MTT實(shí)驗(yàn)檢測細(xì)胞增殖活性；體外劃痕實(shí)驗(yàn)和transwell遷移實(shí)驗(yàn)檢測細(xì)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(dá)；GST-pulldown技術(shù)檢測GTP-Racl表達(dá)。結(jié)果顯示：敲低AMPKal后,miR-451對(duì)細(xì)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(xì)胞瘤腫瘤組織標(biāo)本的miR-451的表達(dá)量顯著低于對(duì)照腦組織標(biāo)本。體外實(shí)驗(yàn)證實(shí),miR-451增強(qiáng)膠質(zhì)瘤細(xì)胞的增殖能力,同時(shí)抑制膠質(zhì)瘤細(xì)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(xì)胞的增殖能力和遷移運(yùn)動(dòng)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(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.41

【引證文獻(xiàn)】

相關(guān)碩士學(xué)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(xué);2016年

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本文編號(hào)：1822155