SENP1对急性髓细胞白血病生物学特征和预后的影响及机制研究

发布时间：2018-02-02 08:18

  本文关键词： 急性髓细胞白血病 SENP1 HOXA9 ZNF521 CD56 CD11b　出处：《第三军医大学》2015年博士论文　论文类型：学位论文

【摘要】：背景和目的:急性髓细胞白血病(acute myeloid leukemia,AML)是我国白血病最常见的类型,发病率约十万分之四,并随着年龄的增长发病率逐年上升。该病以骨髓、外周血和其他器官中出现大量未成熟的髓系前体细胞,这些髓系前体细胞以增殖失控、分化障碍、凋亡受阻为主要的生物学特征,从多个角度抑制正常的造血分化过程,最终导致贫血、出血、感染等临床表现并最终致死。由于AML的临床异质性,导致不同的AML病人预后各不相同,目前通常采用细胞遗传学的方法预测AML的预后,但发现很多细胞遗传学预后指标相同的病人的预后仍差异很大,因此这一指标远不能满足临床预后精准化判断的需要,因此非常有必要寻找新的具有预后意义的分子标记物。蛋白质翻译后类泛素化修饰(small ubiquitin like modifier,SUMO)是由于其修饰过程简洁而功能广泛受到关注。SUMO化修饰是动态可逆的过程,而SENP1(SUMO specific proteinase 1)是作用最强的去SUMO化修饰蛋白酶。前期研究表明SENP1参与红细胞、T细胞和B细胞的发育过程,提示SENP1在造血系统细胞的分化发育过程中有着关键的作用。但SENP1在AML中的表达水平、生物学作用、机制以及在AML病人中的预后意义目前都是未知的。为了进行以上未知领域的探索,本研究共分三部分进行。第一部分通过RT-PCR、免疫组化和蛋白免疫印迹检测SENP1在AML中的表达情况;通过构建敲低SENP1的AML稳转细胞系,观察SENP1对AML细胞增殖和凋亡的影响;应用Kaplan-Meier分析和相关性分析,研究SENP1对于AML预后判断的意义,并研究其与其他报道的预后分子(CD56,CD11b)之间的相关性。第二部分研究HOXA9的SUMO化修饰对AML生物学特征的影响和ZNF521的SUMO化修饰对AML生物学特征的影响,以解释SENP1影响AML生物学特征的可能机制。第三部分采用meta分析的方法明确CD56和CD11b对于AML的预后意义,为解释SENP1的预后作用提供线索。材料与方法:整个研究采用了标本实验、细胞实验、动物实验、临床资料分析等多种手段。标本实验以前期收集的AML病例标本和增生性骨髓象对照标本为主要的研究对象。细胞实验以常用的代表性AML细胞系(如THP-1,K562,U937)为主要的研究对象。动物实验采用nod/scid小鼠构建aml的模型。临床资料分析主要以公共数据库的资料作为研究对象。1.分别用rt-pcr、免疫组化和蛋白免疫印迹检测senp1蛋白和mrna在aml中的表达情况。2.应用shrna技术构建敲低senp1的aml稳转细胞系,观察senp1对aml细胞增殖和凋亡的影响;采用rna-seq的方法寻找senp1可能的下游基因。3.应用kaplan-meier分析和相关性分析,研究senp1对于aml预后判断的意义,并研究其与其他报道的预后分子(cd56,cd11b)之间的相关性。4.通过免疫共沉淀、点突变、构建稳转细胞系等技术,研究hoxa9和znf521的sumo化修饰对aml生物学特征的影响,以期解释senp1发挥调控aml生物学特征作用的可能机制。5.通过meta分析的方法明确与senp1的表达存在显著相关性的预后分子cd56和cd11b的预后意义,为解释senp1的预后作用提供线索。结果:1.senp1蛋白和mrna在aml中均高表达。2.敲低senp1后,aml细胞系的增殖能力减低、凋亡比例上升。3.senp1高表达与aml病人的不良预后相关,并且senp1和目前已报道的预后分子cd56和cd11b之间存在显著相关性。4.hoxa9能够发生sumo化修饰,并且sumo化修饰缺失(位点突变)后hoxa9的促增殖能力更强。hoxa9的sumo化修饰受到senp1的调控。5.znf521能够发生sumo化修饰,并且sumo化修饰缺失(位点突变)后znf521的促增殖能力更强。znf521的sumo化修饰受到senp1的调控6.通过meta分析的方法明确与senp1表达密切相关的cd56和cd11b都是aml的预后预测分子。结论:1.senp1在aml中高表达,具有促进aml细胞增殖、抑制凋亡的能力。2.senp1对aml生物学特征的影响可能是通过调控hoxa9和znf521的sumo化修饰来实现的。3.与senp1表达密切相关的cd56和cd11b都是aml的预后预测分子,为解释senp1的预后预测作用提供了线索。
[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.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.71

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