發(fā)布時(shí)間：2018-02-02 08:18

  本文關(guān)鍵詞： 急性髓細(xì)胞白血病 SENP1 HOXA9 ZNF521 CD56 CD11b　出處：《第三軍醫(yī)大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景和目的:急性髓細(xì)胞白血病(acute myeloid leukemia,AML)是我國(guó)白血病最常見的類型,發(fā)病率約十萬分之四,并隨著年齡的增長(zhǎng)發(fā)病率逐年上升。該病以骨髓、外周血和其他器官中出現(xiàn)大量未成熟的髓系前體細(xì)胞,這些髓系前體細(xì)胞以增殖失控、分化障礙、凋亡受阻為主要的生物學(xué)特征,從多個(gè)角度抑制正常的造血分化過程,最終導(dǎo)致貧血、出血、感染等臨床表現(xiàn)并最終致死。由于AML的臨床異質(zhì)性,導(dǎo)致不同的AML病人預(yù)后各不相同,目前通常采用細(xì)胞遺傳學(xué)的方法預(yù)測(cè)AML的預(yù)后,但發(fā)現(xiàn)很多細(xì)胞遺傳學(xué)預(yù)后指標(biāo)相同的病人的預(yù)后仍差異很大,因此這一指標(biāo)遠(yuǎn)不能滿足臨床預(yù)后精準(zhǔn)化判斷的需要,因此非常有必要尋找新的具有預(yù)后意義的分子標(biāo)記物。蛋白質(zhì)翻譯后類泛素化修飾(small ubiquitin like modifier,SUMO)是由于其修飾過程簡(jiǎn)潔而功能廣泛受到關(guān)注。SUMO化修飾是動(dòng)態(tài)可逆的過程,而SENP1(SUMO specific proteinase 1)是作用最強(qiáng)的去SUMO化修飾蛋白酶。前期研究表明SENP1參與紅細(xì)胞、T細(xì)胞和B細(xì)胞的發(fā)育過程,提示SENP1在造血系統(tǒng)細(xì)胞的分化發(fā)育過程中有著關(guān)鍵的作用。但SENP1在AML中的表達(dá)水平、生物學(xué)作用、機(jī)制以及在AML病人中的預(yù)后意義目前都是未知的。為了進(jìn)行以上未知領(lǐng)域的探索,本研究共分三部分進(jìn)行。第一部分通過RT-PCR、免疫組化和蛋白免疫印跡檢測(cè)SENP1在AML中的表達(dá)情況;通過構(gòu)建敲低SENP1的AML穩(wěn)轉(zhuǎn)細(xì)胞系,觀察SENP1對(duì)AML細(xì)胞增殖和凋亡的影響;應(yīng)用Kaplan-Meier分析和相關(guān)性分析,研究SENP1對(duì)于AML預(yù)后判斷的意義,并研究其與其他報(bào)道的預(yù)后分子(CD56,CD11b)之間的相關(guān)性。第二部分研究HOXA9的SUMO化修飾對(duì)AML生物學(xué)特征的影響和ZNF521的SUMO化修飾對(duì)AML生物學(xué)特征的影響,以解釋SENP1影響AML生物學(xué)特征的可能機(jī)制。第三部分采用meta分析的方法明確CD56和CD11b對(duì)于AML的預(yù)后意義,為解釋SENP1的預(yù)后作用提供線索。材料與方法:整個(gè)研究采用了標(biāo)本實(shí)驗(yàn)、細(xì)胞實(shí)驗(yàn)、動(dòng)物實(shí)驗(yàn)、臨床資料分析等多種手段。標(biāo)本實(shí)驗(yàn)以前期收集的AML病例標(biāo)本和增生性骨髓象對(duì)照標(biāo)本為主要的研究對(duì)象。細(xì)胞實(shí)驗(yàn)以常用的代表性AML細(xì)胞系(如THP-1,K562,U937)為主要的研究對(duì)象。動(dòng)物實(shí)驗(yàn)采用nod/scid小鼠構(gòu)建aml的模型。臨床資料分析主要以公共數(shù)據(jù)庫的資料作為研究對(duì)象。1.分別用rt-pcr、免疫組化和蛋白免疫印跡檢測(cè)senp1蛋白和mrna在aml中的表達(dá)情況。2.應(yīng)用shrna技術(shù)構(gòu)建敲低senp1的aml穩(wěn)轉(zhuǎn)細(xì)胞系,觀察senp1對(duì)aml細(xì)胞增殖和凋亡的影響;采用rna-seq的方法尋找senp1可能的下游基因。3.應(yīng)用kaplan-meier分析和相關(guān)性分析,研究senp1對(duì)于aml預(yù)后判斷的意義,并研究其與其他報(bào)道的預(yù)后分子(cd56,cd11b)之間的相關(guān)性。4.通過免疫共沉淀、點(diǎn)突變、構(gòu)建穩(wěn)轉(zhuǎn)細(xì)胞系等技術(shù),研究hoxa9和znf521的sumo化修飾對(duì)aml生物學(xué)特征的影響,以期解釋senp1發(fā)揮調(diào)控aml生物學(xué)特征作用的可能機(jī)制。5.通過meta分析的方法明確與senp1的表達(dá)存在顯著相關(guān)性的預(yù)后分子cd56和cd11b的預(yù)后意義,為解釋senp1的預(yù)后作用提供線索。結(jié)果:1.senp1蛋白和mrna在aml中均高表達(dá)。2.敲低senp1后,aml細(xì)胞系的增殖能力減低、凋亡比例上升。3.senp1高表達(dá)與aml病人的不良預(yù)后相關(guān),并且senp1和目前已報(bào)道的預(yù)后分子cd56和cd11b之間存在顯著相關(guān)性。4.hoxa9能夠發(fā)生sumo化修飾,并且sumo化修飾缺失(位點(diǎn)突變)后hoxa9的促增殖能力更強(qiáng)。hoxa9的sumo化修飾受到senp1的調(diào)控。5.znf521能夠發(fā)生sumo化修飾,并且sumo化修飾缺失(位點(diǎn)突變)后znf521的促增殖能力更強(qiáng)。znf521的sumo化修飾受到senp1的調(diào)控6.通過meta分析的方法明確與senp1表達(dá)密切相關(guān)的cd56和cd11b都是aml的預(yù)后預(yù)測(cè)分子。結(jié)論:1.senp1在aml中高表達(dá),具有促進(jìn)aml細(xì)胞增殖、抑制凋亡的能力。2.senp1對(duì)aml生物學(xué)特征的影響可能是通過調(diào)控hoxa9和znf521的sumo化修飾來實(shí)現(xiàn)的。3.與senp1表達(dá)密切相關(guān)的cd56和cd11b都是aml的預(yù)后預(yù)測(cè)分子,為解釋senp1的預(yù)后預(yù)測(cè)作用提供了線索。
[Abstract]:Background and objective: acute myeloid leukemia (acute myeloid, leukemia, AML) is the most common type of leukemia, the incidence rate of about 4/100000, and with the age growth rate increased year by year. The disease in bone marrow, peripheral blood and other organs without the emergence of a large number of mature myeloid precursor cells, these myeloid precursor cells to proliferation, differentiation disorders, apoptosis as the main biological characteristics of the inhibition of normal hematopoietic differentiation from multiple perspectives, eventually leading to anemia, bleeding, infection and other clinical manifestations and eventually death. Because of the clinical heterogeneity of AML, AML in the prognosis of the patients with different, usually at present by using the method of cytogenetics in predicting the prognosis of AML patients, but many cytogenetic prognostic indicators of the same is different, so this index can not meet the clinical pre accurate judgment Need, it is very necessary to find out has prognostic significance of new molecular markers. Ubiquitin protein post-translational modification type (small ubiquitin like modifier, SUMO) is due to the modification process is simple and functional modification of.SUMO is reversible and dynamic process, SENP1 (SUMO specific proteinase 1) is the strongest go to the modification of SUMO protease. Previous studies indicated that SENP1 participate in the development process of red blood cells, T cells and B cells, suggesting that SENP1 plays a pivotal role in differentiation in hematopoietic cell development process. But the expression level of SENP1 AML in the biological effects, mechanism and prognostic significance in patients with AML are currently in order to explore the unknown. Above the unknown, this study is divided into three parts. The first part by RT-PCR, expression of SENP1 by immunohistochemistry and Western blot in AML Condition; by constructing the knockdown of SENP1 AML stably transfected cell lines, the effect of SENP1 on proliferation and apoptosis of AML cells; the application of Kaplan-Meier analysis and correlation analysis, the research of SENP1 for AML prognosis, and the study and other reports (CD56, CD11b) molecular prognostic correlation between the second part of the HOXA9. The SUMO modification effect on the biological characteristics of AML and ZNF521 SUMO modification effect on the biological characteristics of AML, to interpret the possible mechanism of SENP1 influence the biological characteristics of AML. The third part uses meta analysis method to clear CD56 and CD11b on the prognostic significance of AML, provide clues for the prognostic role of SENP1. Materials and methods the study uses: specimens, cell experiment, animal experiment, various means of clinical data analysis. Specimens collected earlier experiments with AML cases and bone marrow hyperplasia of specimens The main object of study. Cell experiments with common representative AML cell lines (such as THP-1, K562, U937) as the main research object. Animal experiments using nod/scid mice to construct the AML model. The clinical data analysis on the public database as the research object.1. respectively with RT-PCR,.2. constructed by shRNA technology to knockdown SENP1 AML of stabletransfection cell lines expression by immunohistochemistry and Western blot detection of SENP1 protein and mRNA in AML, the effect of SENP1 on proliferation and apoptosis of AML cells; using RNA-seq method to find SENP1 downstream genes possible application of.3. analysis Kaplan-Meier analysis and correlation study of SENP1 for AML and prognosis. The study and other reports (CD56, CD11b) molecular prognostic correlation between.4. by CO immunoprecipitation, point mutation, construct stabletransfection cell lines of HOXA9 and znf521 technology, sumo Effect of modification on AML biological characteristics, in order to explain the possible mechanism of.5. SENP1 regulate the biological characteristics of AML function through the method of meta analysis clearly correlated with the prognostic significance of SENP1 expression in the prognosis of molecules CD56 and CD11b, provide clues for the prognostic role of SENP1. Results: the expression of 1.senp1 and mRNA were highly expressed in AML.2. knockdown of SENP1, reduce the AML cell proliferation, apoptosis increased the proportion of poor prognosis of patients with AML high expression of.3.senp1 and SENP1 and the correlation between the prognosis of molecules reported in CD56 and CD11b to SUMO modification occurred in significant correlation between.4.hoxa9 and sumo, modification deletion (mutation) after HOXA9 the proliferation ability of.Hoxa9 was stronger SUMO modification by regulation of.5.znf521 SENP1 can happen SUMO modification, SUMO modification and deletion (mutation) after znf521 The proliferation of stronger.Znf521 SUMO modification method regulated by SENP1 6. through meta analysis clearly and the expression of SENP1 is closely related to the CD56 and CD11b molecules are predicting the prognosis of AML. Conclusion: the high expression of 1.senp1 in AML, with the promotion of AML cell proliferation, inhibition of apoptosis can influence on the biological characteristics of AML.2.senp1.3. and SENP1 may be achieved through modification of SUMO regulation of HOXA9 and znf521 is closely related with the expression of CD56 and CD11b molecules are predicting the prognosis of AML, provides clues for the interpretation of SENP1 prognosis prediction.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R733.71

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