本文選題：急性髓系白血病 + microRNA　； 參考：《浙江大學(xué)》2017年博士論文

【摘要】：背景:急性髓系白血病(acutemyeloid leukemia,AML)是目前成人最常見(jiàn)的血液系統(tǒng)惡性腫瘤之一,在細(xì)胞形態(tài)學(xué)、分子生物學(xué)、細(xì)胞遺傳學(xué)、免疫分型、臨床表現(xiàn)等方面具有高度異質(zhì)性。AML的發(fā)病機(jī)制十分復(fù)雜,到目前為止,科學(xué)家們還不能完全將之闡述清楚�，F(xiàn)在公認(rèn)對(duì)疾病發(fā)生發(fā)展有重要啟動(dòng)作用的包括染色體核型異常和可重現(xiàn)性的基因突變。通過(guò)對(duì)這兩個(gè)因素的分類,可以對(duì)大部分病人進(jìn)行危險(xiǎn)分層,根據(jù)病人不同的危險(xiǎn)分層,采用相應(yīng)的治療方案,使得AML患者的五年生存率有顯著的提高。但是,部分患者預(yù)后仍然不佳,提示臨床醫(yī)生需要進(jìn)一步探索AML新的發(fā)病機(jī)制,完善現(xiàn)有的預(yù)后評(píng)估系統(tǒng)。因此,迫切需要發(fā)現(xiàn)新的預(yù)后因子并探討這些預(yù)后因子在AML中發(fā)揮的功能,從而完善現(xiàn)有的評(píng)估系統(tǒng)。近年來(lái),隨著科學(xué)家們對(duì)表觀遺傳學(xué)認(rèn)識(shí)的不斷深入,越來(lái)越多的證據(jù)表明其在AML發(fā)生發(fā)展中發(fā)揮著重要的作用。microRNAs(miRNAs)作為表觀遺傳學(xué)中重要的一員,在AML中的作用正越來(lái)越受到重視起來(lái)。MiRNAs是一類長(zhǎng)度為18-25個(gè)核苷酸,具有在轉(zhuǎn)錄后調(diào)控基因表達(dá)水平的非編碼小RNA,可通過(guò)特異性結(jié)合靶基因的3'非翻譯區(qū)(3'UTR)上的互補(bǔ)序列,從而下調(diào)靶基因的表達(dá)水平。目前已有很多文獻(xiàn)表明miRNAs的表達(dá)異常和AML的發(fā)生發(fā)展有著密切的聯(lián)系。目的:收集AML患者和正常對(duì)照的標(biāo)本,利用熒光定量PCR檢測(cè)miR-320c的表達(dá)情況,分析miR-320c的表達(dá)和AML患者預(yù)后之間的關(guān)系,探索miR-320c表達(dá)下調(diào)的調(diào)控機(jī)制,采用獲得性功能實(shí)驗(yàn)觀察miR-320c在AML細(xì)胞株中的生物學(xué)功能,利用生物信息學(xué)網(wǎng)站和熒光素酶雙報(bào)告系統(tǒng)尋找miR-320c下游直接作用的靶基因,并進(jìn)一步分析靶基因?qū)ML患者預(yù)后的影響、對(duì)AML細(xì)胞株生物學(xué)表型的調(diào)節(jié)以及對(duì)下游信號(hào)通路的調(diào)控。方法:(1)利用定量PCR手段檢測(cè)304例AML患者及正常對(duì)照中miR-320c的表達(dá)水平,統(tǒng)計(jì)學(xué)分析miR-320c表達(dá)和AML患者預(yù)后之間的關(guān)系;(2)探討miR-320c低表達(dá)是否和其啟動(dòng)子區(qū)CpG島高度甲基化相關(guān);通過(guò)轉(zhuǎn)染miR-320c模擬體(mimic)外源性過(guò)表達(dá)miR-320c來(lái)進(jìn)行獲得性功能實(shí)驗(yàn),通過(guò)MTS、集落形成實(shí)驗(yàn)、流式檢測(cè)細(xì)胞凋亡和細(xì)胞周期、WfesternBlot檢測(cè)相關(guān)蛋白的改變等來(lái)探索miR-320c在AML中的生物學(xué)功能;利用生物信息學(xué)網(wǎng)站對(duì)miR-320c的靶基因進(jìn)行預(yù)測(cè),通過(guò)熒光定量PCR和WesternBlot檢測(cè)靶基因RNA及蛋白水平的改變,熒光素酶雙報(bào)告實(shí)驗(yàn)驗(yàn)證miR-320c和靶基因之間的關(guān)系;(3)檢測(cè)304例AML患者及正常對(duì)照中靶基因的表達(dá)水平,分析靶基因表達(dá)和AML患者預(yù)后之間的關(guān)系;干擾靶基因表達(dá),模擬miR-320c過(guò)表達(dá)在AML中發(fā)揮的作用,探索靶基因的生物學(xué)功能,明確靶向調(diào)控關(guān)系及其下游調(diào)控機(jī)制。結(jié)果:(1)和正常對(duì)照相比miR-320c在AML患者中表達(dá)顯著下調(diào),miR-320c低表達(dá)患者的OS較差,miR-320c高表達(dá)水平是AML患者的預(yù)后良好的獨(dú)立因素;(2)去甲基化藥物地西他濱處理AML細(xì)胞株THP-1、MV4-11和AML-OCI3后,可誘導(dǎo)miR-320c的表達(dá)上調(diào);(3)過(guò)表達(dá)miR-320c可以通過(guò)PI3K/AKT及凋亡信號(hào)通路抑制AML的細(xì)胞活性、增殖及克隆形成能力,誘導(dǎo)細(xì)胞凋亡;(4)過(guò)表達(dá)miR-320c可以增加AML細(xì)胞對(duì)阿糖胞苷的敏感性;(5)利用生物信息學(xué)網(wǎng)站預(yù)測(cè)Musashi2(MSI2)是miR-320c的靶基因,并通過(guò)熒光定量PCR、WeesternBlot實(shí)驗(yàn)和熒光素酶雙報(bào)告系統(tǒng)證實(shí)MSI2是miR-320c的下游靶基因;(6)和正常對(duì)照相比,MSI2在AML患者中表達(dá)上調(diào),MSI2高表達(dá)患者年齡較大,NPM1和FLT3-ITD的突變率較高,CR率較低,OS和EFS較差;(7)慢病毒干擾MSI2的表達(dá)可以抑制AML細(xì)胞株的增殖,誘導(dǎo)細(xì)胞凋亡,并且可以下調(diào)MAPK及WNT通路。結(jié)論:(1)miR-320c在AML患者中顯著低表達(dá),miR-320c高表達(dá)水平是AML患者的預(yù)后良好的獨(dú)立因素,低表達(dá)患者預(yù)后不良;(2)miR-320C低表達(dá)和其啟動(dòng)子區(qū)CpG島高甲基化相關(guān);(3)過(guò)表達(dá)miR-320c可以抑制AML細(xì)胞活性、阻礙細(xì)胞增殖、降低克隆形成能力,誘導(dǎo)細(xì)胞凋亡,下調(diào)PI3K/AKT通路活性,增加對(duì)阿糖胞苷的敏感性;(4)MSI2是miR-320c的直接靶點(diǎn),MSI2在AML患者中高表達(dá),MSI2過(guò)表達(dá)是AML患者的預(yù)后不良因素;(5)下調(diào)MSI2可以抑制AML細(xì)胞的增殖,誘導(dǎo)細(xì)胞凋亡,下調(diào)細(xì)胞內(nèi)MAPK和WNT信號(hào)通路活性。
[Abstract]:Background: acutemyeloid leukemia (AML) is one of the most common hematological malignancies in adults. The pathogenesis of highly heterogeneous.AML in cellular morphology, molecular biology, cytogenetics, immunophenotype and clinical manifestations is very complicated. So far, scientists can not completely It is clear. It is now recognized that the genetic abnormalities and reproducibility of chromosomes are important for the development of disease. By classifying these two factors, the risk stratification of most patients can be stratified, according to the different risk stratification of the patients, and the corresponding treatment scheme is adopted to make the AML patients five years old. There is a significant improvement in the survival rate. However, the prognosis of some patients is still poor, suggesting that clinicians need to further explore the new pathogenesis of AML and improve the existing prognostic evaluation system. Therefore, there is an urgent need to discover new prognostic factors and explore the function of these prognostic factors in AML so as to improve the existing evaluation system. As scientists continue to understand epigenetics, more and more evidence suggests that.MicroRNAs plays an important role in the development of AML (miRNAs) as an important member of epigenetics. The role in AML is becoming more and more important, and.MiRNAs is a class of 18-25 nucleotides, which is in transcription. The non coding small RNA of the post regulatory gene expression level can be regulated by a complementary sequence on the 3'non translation region (3'UTR) that specifically combines the target gene, so that the expression level of the target gene is down regulated. Many documents have shown that there is a close relationship between the abnormal expression of miRNAs and the development of AML. Objective: to collect the specimens of the AML patients and the normal controls. The expression of miR-320c was detected by fluorescence quantitative PCR, the relationship between the expression of miR-320c and the prognosis of AML patients was analyzed. The regulation mechanism of the down regulation of miR-320c expression was explored. The biological function of miR-320c in the AML cell line was observed by the acquired functional experiment, and the bioinformatics website and the luciferase double report system were used to find miR-320. The direct target gene of C downstream, and further analysis of the effect of target gene on the prognosis of AML patients, the regulation of the biological phenotype of the AML cell line and the regulation of the downstream signal pathway. Methods: (1) the expression level of miR-320c in 304 cases of AML patients and normal controls was detected by quantitative PCR, and miR-320c expression and AML patients were statistically analyzed. The relationship between the prognosis; (2) to investigate whether the low expression of miR-320c is related to the high methylation of CpG island in the promoter region; through transfection of miR-320c analogue (mimic) exogenous overexpression of miR-320c for acquired functional experiments, MTS, colony formation experiments, flow detection of apoptosis and cell cycle, and the modification of WfesternBlot detection related proteins To explore the biological function of miR-320c in AML, to predict the target gene of miR-320c by bioinformatics website, to detect the change of target gene RNA and protein level by fluorescence quantitative PCR and WesternBlot, and to verify the relationship between miR-320c and the target gene by the double report of luciferase reporter; (3) to detect 304 cases of AML patients and normal pairs. According to the expression level of the target gene, the relationship between the target gene expression and the prognosis of AML patients was analyzed, the target gene expression, the role of miR-320c overexpression in AML were simulated, the biological function of the target gene, the target regulation relationship and the downstream regulation mechanism were explored. Results: (1) and the normal contrast miR-320c was expressed in AML patients. Significant downregulation, low expression of OS in patients with miR-320c low expression, high expression of miR-320c is an independent factor for good prognosis in AML patients; (2) the expression of miR-320c can be induced after AML cell line THP-1, MV4-11 and AML-OCI3 of demethylation drug, and (3) miR-320c can inhibit AML by PI3K/AKT and apoptotic signaling pathways. Cell activity, proliferation and clone formation ability, inducing cell apoptosis; (4) overexpression of miR-320c can increase the sensitivity of AML cells to cytarabine; (5) using bioinformatics website to predict Musashi2 (MSI2) is the target gene of miR-320c, and through fluorescence quantitative PCR, WeesternBlot experiment and luciferase double report system confirmed MSI2 is miR-320c (6) compared with normal control, the expression of MSI2 in AML patients was up-regulated, MSI2 high expression was older, NPM1 and FLT3-ITD had higher mutation rate, CR rate was lower, OS and EFS were poor; (7) the expression of lentivirus interfering MSI2 could inhibit the proliferation of AML cell lines, induce apoptosis and down regulation of MAPK and WNT pathways. Conclusion: (1) IR-320c was significantly lower expression in AML patients. High expression of miR-320c was an independent factor in the prognosis of AML patients, and low expression of patients had poor prognosis; (2) low expression of miR-320C was associated with CpG island Gao Jiaji in promoter region; (3) overexpression of miR-320c could inhibit the viability of AML cells, impede cell proliferation, and reduce the ability to clone and induce cells to induce cells. Apoptosis, down-regulation of PI3K/AKT pathway activity, increase the sensitivity of cytarabine; (4) MSI2 is a direct target of miR-320c, MSI2 is highly expressed in AML patients. MSI2 overexpression is a prognostic factor for AML patients; (5) down regulation of MSI2 can inhibit the proliferation of AML cells, induce apoptosis, and down regulate the activity of MAPK and WNT signaling pathway in cells.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R733.71

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王前;骨髓增生異常綜合征轉(zhuǎn)化為急性髓系白血病1例[J];白血病.淋巴瘤;2001年06期

2 溫莉娟,孟昭彥,劉利明,侯美仙,陳麗燕;染色體t(8;21)伴急性髓系白血病六例[J];中華醫(yī)學(xué)遺傳學(xué)雜志;2004年04期

3 徐升,李麗,朱子玲,張日;血管內(nèi)皮生長(zhǎng)因子在急性髓系白血病中的表達(dá)[J];中國(guó)血液流變學(xué)雜志;2004年03期

4 上海市中美白血病合作課題組;微分化急性髓系白血病四例報(bào)告[J];中華血液學(xué)雜志;2005年06期

5 夏云金,萬(wàn)楚成,郭仁慈;不同病期急性髓系白血病患者紅細(xì)胞免疫功能檢測(cè)及臨床意義[J];微循環(huán)學(xué)雜志;2005年02期

6 李玉芹;戴瓊;龔武清;楊曉秋;;微分化急性髓系白血病1例[J];淮海醫(yī)藥;2007年04期

7 劉凱奇;秘營(yíng)昌;李大鵬;白潔;井麗萍;卞壽庚;王建祥;;急性髓系白血病合并慢性淋巴細(xì)胞白血病一例報(bào)道及文獻(xiàn)復(fù)習(xí)[J];繼續(xù)醫(yī)學(xué)教育;2007年04期

8 謝曉恬;;兒童急性髓系白血病的診斷與治療[J];實(shí)用兒科臨床雜志;2011年15期

9 ;探尋白血病線索[J];世界科學(xué);2013年08期

10 肖志堅(jiān)，，王建祥，郝玉書，李建波，陳佩貞，卞壽庚，錢林生，孟慶祥;兩例無(wú)t（8；21）M_（2b）型急性髓系白血病的基因檢測(cè)[J];中華醫(yī)學(xué)遺傳學(xué)雜志;1994年05期

相關(guān)會(huì)議論文 前10條

1 李達(dá);葛志紅;吳順杰;梁冰;;白血康為主治療難治性復(fù)發(fā)急性髓系白血病12例臨床分析[A];第六屆全國(guó)中西醫(yī)結(jié)合血液病學(xué)術(shù)會(huì)議論文匯編[C];2002年

2 范洵楠;邵曉雁;楊永公;陳兵;許景艷;孫雪梅;歐陽(yáng)建;;預(yù)激療法治療難治性或復(fù)發(fā)性急性髓系白血病的療效分析[A];2005年華東六省一市血液病學(xué)學(xué)術(shù)會(huì)議暨浙江省血液病學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2005年

3 華佳葉;龐纓;蔡曉東;葉絮;馮瑩;;急性髓系白血病自體造血干細(xì)胞移植后的維持化療[A];第11次中國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文匯編[C];2007年

4 王建祥;;急性髓系白血病的合理治療[A];2011年浙江省血液病學(xué)術(shù)年會(huì)暨浙江省醫(yī)學(xué)會(huì)血液病學(xué)分會(huì)成立50周年慶典論文匯編[C];2011年

5 楊文華;;急性髓系白血病中西醫(yī)結(jié)合治療策略[A];2009年浙江省中醫(yī)藥學(xué)會(huì)血液病學(xué)術(shù)年會(huì)、浙江省中西醫(yī)結(jié)合學(xué)會(huì)血液病學(xué)術(shù)年會(huì)暨國(guó)家級(jí)中西醫(yī)結(jié)合血液病新進(jìn)展繼續(xù)教育學(xué)習(xí)班論文匯編[C];2009年

6 顧龍君;帖利軍;陳靜;潘慈;董璐;陳靜;葉輝;薛惠良;湯靜燕;王耀平;鄒佳音;;兒童急性髓系白血病預(yù)后因素分析[A];中華醫(yī)學(xué)會(huì)第八次全國(guó)血液學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2004年

7 張曉燕;李軍民;;急性髓系白血病染色體異常與療效關(guān)系[A];中華醫(yī)學(xué)會(huì)第八次全國(guó)血液學(xué)學(xué)術(shù)會(huì)議論文匯編[C];2004年

8 陳賽娟;王月英;王蘭;周光飚;梁洋;焦波;武傳鳳;陳竺;;T(8；21)急性髓系白血病多步驟發(fā)病機(jī)制和靶向治療研究[A];第11次中國(guó)實(shí)驗(yàn)血液學(xué)會(huì)議論文匯編[C];2007年

9 金潔;;急性髓系白血病的治療進(jìn)展[A];2007年浙江省血液病學(xué)術(shù)年會(huì)論文匯編[C];2007年

10 金潔;;急性髓系白血病的治療進(jìn)展[A];第七屆全國(guó)血液免疫學(xué)學(xué)術(shù)大會(huì)暨2008年浙江省血液病學(xué)術(shù)年會(huì)論文匯編[C];2008年

相關(guān)重要報(bào)紙文章 前3條

1 中國(guó)醫(yī)學(xué)科學(xué)院血液學(xué)研究所 王迎 王建祥;急性髓系白血病未解難題仍很多[N];健康報(bào);2013年

2 記者 馮衛(wèi)東;加找到急性髓系白血病復(fù)發(fā)根源[N];科技日?qǐng)?bào);2014年

3 通訊員 章巍 記者 程守勤;漢黃芩苷或可治療急性髓系白血病[N];健康報(bào);2013年

相關(guān)博士學(xué)位論文 前10條

1 丁超;核心結(jié)合因子相關(guān)急性髓系白血病的細(xì)胞和分子遺傳學(xué)異常[D];蘇州大學(xué);2015年

2 尹甲偉;BCL11A在中國(guó)急性髓系白血病病人中的表達(dá)水平及其調(diào)控機(jī)制研究[D];蘇州大學(xué);2015年

3 王凌波;聚二磷酸腺苷核糖聚合酶1在急性髓系白血病中的作用及機(jī)制研究[D];山東大學(xué);2016年

4 許華;SIRT2在急性髓系白血病多藥耐藥中的作用研究[D];北京協(xié)和醫(yī)學(xué)院;2016年

5 余國(guó)攀;AML1/ETO陽(yáng)性急性髓系白血病的分子生物學(xué)特征與分層診斷體系的建立[D];南方醫(yī)科大學(xué);2016年

6 陳曉平;地塞米松抑制t(8;21)急性髓系白血病細(xì)胞增殖的研究[D];中國(guó)人民解放軍醫(yī)學(xué)院;2016年

7 王利軍;Chidamide對(duì)急性髓系白血病作用及相關(guān)機(jī)制的研究[D];中國(guó)人民解放軍醫(yī)學(xué)院;2016年

8 胡超;miR-550-1在急性髓系白血病中的抑癌作用及其機(jī)制研究[D];浙江大學(xué);2016年

9 高娜;急性髓系白血病Galectin-3表達(dá)及其意義[D];山東大學(xué);2016年

10 余夢(mèng)霞;miR-320c在急性髓系白血病中的臨床意義及其機(jī)制研究[D];浙江大學(xué);2017年

相關(guān)碩士學(xué)位論文 前10條

1 肖世極;EVI1基因在成人急性髓系白血病的預(yù)后分析[D];福建醫(yī)科大學(xué);2015年

2 郝其姍;1.t(1;9)(p22;q34)形成DEK/NUP214的急性髓系白血病一例報(bào)告并文獻(xiàn)復(fù)習(xí) 2.急性髓系白血病IGF2BPs和Nek2的表達(dá)及預(yù)后研究[D];北京協(xié)和醫(yī)學(xué)院;2015年

3 矯麗麗;IA方案在急性髓系白血病療效及副反應(yīng)的臨床研究[D];泰山醫(yī)學(xué)院;2014年

4 耿麗;MTT法體外藥敏試驗(yàn)在優(yōu)化急性髓系白血病治療方案中的應(yīng)用[D];河北醫(yī)科大學(xué);2015年

5 朱穎超;FLT3-ITD基因突變急性髓系白血病的臨床及實(shí)驗(yàn)室特征分析[D];鄭州大學(xué);2015年

6 謝惠敏;t(8;21)急性髓系白血病患者FLT3基因高表達(dá)的臨床意義及機(jī)制研究[D];中國(guó)人民解放軍醫(yī)學(xué)院;2015年

7 鄭慧菲;DNA甲基化在急性髓系白血病和骨髓增生異常綜合征中的應(yīng)用[D];蘇州大學(xué);2015年

8 丁莎莎;FLT3-ITD突變數(shù)量、長(zhǎng)度及水平對(duì)急性髓系白血病患者預(yù)后的影響[D];蘇州大學(xué);2015年

9 馬金鳳;Tim-3在急性髓系白血病NK細(xì)胞上表達(dá)特征及臨床意義[D];蘇州大學(xué);2015年

10 汪生;異基因造血干細(xì)胞移植治療AML1-ETO陽(yáng)性急性髓系白血病M2亞型的療效分析[D];安徽醫(yī)科大學(xué);2015年

本文編號(hào)：1963775