Aurora家族基因在急性白血病中的表达及其临床意义

发布时间：2018-03-22 17:47

  本文选题：Aurora基因　切入点：急性白血病　出处：《郑州大学》2017年硕士论文　论文类型：学位论文

【摘要】：Aurora家族基因包括Aurora-A、B、C,它们表达的蛋白是一组高度相关的Aurora激酶家族,在正常细胞中对调控细胞分裂起着至关重要的作用,Aurora-A激酶的主要功能是中心体的调节和有丝分裂纺锤体的形成,Aurora-B激酶是一种旅客蛋白,其功能主要是确保染色体的分离和胞质分裂,Aurora-C激酶主要在减数分裂中起重要作用,也有研究显示Aurora-C激酶也是一种旅客蛋白,对Aurora-B激酶的功能起补充作用,Aurora激酶的失调或异常会引起细胞分裂异常,进而促使染色体不稳定和肿瘤的形成。从目前研究来看,Aurora-C激酶主要参与生精过程,对有丝分裂特别是对中心体的功能我们了解的还不够深入,尤其是其对肿瘤发生、发展、耐药的机制及意义,有研究认为其对肿瘤不起作用,有研究认为其促进Aurora-B激酶的功能,有研究认为Aurora-C激酶也能促进细胞转化和肿瘤形成。在多种肿瘤中Aurora-A和B激酶高表达,其表达异常能够激活调节细胞增殖的相关信号传导通路,阻碍细胞分化与凋亡,其表达水平和肿瘤的分期、预后等都有一定的相关性,然而,Aurora基因与急性白血病(AL)临床指标的关系鲜有研究,所以Aurora-A、B、C mRNA表达水平与急性白血病类型、初治白血病细胞(WBC)水平、乳酸脱氢酶(LDH)水平、危险分层及预后不良表面抗原(CD34、CD56、CD71)等临床指标的关系将在本实验中要进一步研究。研究目的本研究中初步采用荧光定量PCR对初诊和化疗完全缓解的AL病人与健康志愿者骨髓单个核细胞中AUR-A、B、C mRNA表达水平进行检测,并探讨其与AL临床指标的关系及3基因表达水平之间的相关性。材料与方法收集2016年5月至9月至郑州大学第一附属医院血液科住院并考虑AL患者和已明确诊断为AL来院强化治疗患者以及健康志愿者的骨髓标本2ml,每个标本进行编号,搜集患者姓名、住院号、标本日期与编号相对应。应用淋巴细胞分离液分离骨髓单个核细胞,应用Trizol提取骨髓单个核细胞总RNA,琼脂糖电泳鉴定RNA完整性,紫外分光光度计检测RNA纯度和浓度,舍弃RNA质量不合格(RNA不完整,不纯或者浓度过低)的标本,质量合格标本加入RNA酶抑制剂并应用无RNA酶水调整RNA浓度至400ng/μL,然后暂时存放到-80℃冰箱保存。初诊未明确诊断患者明确诊断后剔除非急性白血病标本,收集剩余标本的临床资料如性别、年龄、急性白血病分型(急性髓系白血病M0、M1、M2、M3、M4、M5、M6、M7,急性T或B淋巴细胞白血病)、初诊WBC计数、幼稚细胞比例、初诊LDH水平、染色体类型(正常核型、t(15;17)、t(8;21)、t(9;22)、复杂核型、其它)、危险分层(低危、中危、高危)、化疗缓解情况(1疗程缓解与否)、基因突变情况和与预后相关的细胞CD分子阳性百分比(CD34、CD56、CD71),剔除临床资料不完整的标本,符合条件的标本应用荧光定量PCR检测Aurora-A、B、C mRNA相对表达量,其等于2-ΔCT,其中ΔCT=目的基因CT-内参基因CT,内参基因选用GAPDH。应用SPSS 17.0软件对数据进行统计分析,对2组间表达差异采用非参数检验2个独立样本检验,对于多组间表达差异采用非参数检验k个独立样本检验,组间差异有统计学意义者采用Bonferroni法进行多重比较,P0.05表示差异有统计学意义,对于定量资料的相关性分析采用Spearman相关性分析。结果1 Aurora家族基因在不同分组中的表达差异Aurora-A、B、C基因mRNA表达水平在73例初诊AL患者均高于14名健康志愿者和20例化疗完全缓解AL患者(P0.01),而3种基因mRNA表达水平在健康组和缓解组差异均无统计学意义(P0.05),在急性淋巴细胞白血病(ALL)组均高于急性髓系白血病(AML)组(P0.01)。Aurora-A、B、C基因mRNA表达在CD34、CD71、CD56阴性与阳性组差异无统计学意义(P0.05),3种基因表达在不同性别组和1疗程化疗缓解与未缓解组差异均无统计学意义(P0.05)。Aurora-A、B、C表达在不同染色体分组中差异无统计学意义(P0.05)。AUR-A、B、C mRNA表达在高危组均高于低危组(P0.05),在中危组和低危组与高危组和中危组Aurora-A、B、C表达差异均无统计学意义(P0.05)。2 Aurora家族基因与临床指标的相关性73例初诊AL患者中,Aurora-A mRNA表达水平与初诊LDH水平和危险分层呈显著正相关(r=0.279,P=0.017;r=0.314,P=0.007);Aurora-B与初诊LDH水平和危险分层呈显著正相关(r=0.277,P=0.018;r=0.349,P=0.002),Aurora-A、B均与年龄、初诊WBC水平、初诊骨髓幼稚细胞比例和1疗程是否缓解无显著相关性;Aurora-C表达水平与初诊WBC水平、初诊LDH水平和危险分层呈显著正相关(r=0.263,P=0.025;r=0.348,P=0.003;r=0.376,P=0.001),与年龄在0.05水平显著负相关(r=-0.241,P=0.040),而与初诊骨髓中幼稚细胞比例和1疗程是否缓解无显著相关性。3 Aurora家族基因之间表达水平的相关性Aurora-A和B(r=0.444,P=0.000)、Aurora-B和C(r=0.763,P=0.000)、Aurora-A和C(r=0.616,P=0.000)mRNA表达水平均呈显著正相关。结论1.Aurora-A、B、C mRNA在初诊AL中相对于健康人和化疗缓解患者高表达,且在ALL中高于AML,在T-ALL高于B-ALL,提示,Aurora家族基因可能均参与了AL的发生发展,并可以作为AL预后的标志物。2.Aurora-A、B、C mRNA表达水平与初诊WBC水平、LDH水平和危险分层显著正相关,提示Aurora家族基因均可以作为危险分层或预后的指标。3.Aurora-A、B、C mRNA表达水平在AL中显著正相关,提示3基因在AL的发生、发展过程中可能存在功能互补或重叠。
[Abstract]:The Aurora family genes including Aurora-A, B, C, which is a group of highly expressed protein Aurora related kinase family in normal cells, regulation of cell division plays a vital role, the main function of Aurora-A kinase is centrosome regulation and mitotic spindle formation, Aurora-B kinase is a protein of the passenger. The main function is to ensure that chromosome segregation and cytokinesis, Aurora-C kinase mainly plays an important role in meiosis, it has been shown that Aurora-C kinase is a protein supplement for passengers, the function of Aurora-B kinase, Aurora kinase disorder or abnormal will cause abnormal cell division, and promote the formation of chromosome instability and cancer from the present study, Aurora-C kinase involved in spermatogenesis, especially on mitotic centrosome function of our understanding is not deep enough, especially The tumorigenesis, development, mechanism and significance of multidrug resistance, studies suggest that the tumor does not work, have suggested that the promotion of the function of Aurora-B kinase, studies suggest that Aurora-C kinase can also promote cell transformation and tumor formation. The high expression of Aurora-A in tumors and B kinase, its abnormal expression can the activation of related signal transduction pathways regulating cell proliferation, differentiation and apoptosis of cell block, its expression level and tumor staging, prognosis has certain relevance, however, Aurora gene and acute leukemia (AL) relationship between clinical indicators so little research, Aurora-A, B, C mRNA expression level and early acute leukemia. The treatment of leukemia cells (WBC), lactate dehydrogenase (LDH) level, risk stratification and prognosis of surface antigens (CD34, CD56, CD71) the relationship between clinical indicators will be further studied in this experiment. The purpose of the study AUR-A, fluorescence quantitative PCR in complete remission and chemotherapy for newly diagnosed AL patients and healthy volunteers in the bone marrow mononuclear cells was used in this study B, C mRNA expression levels were detected, and the correlation of AL and explore their relationship with clinical indicators and 3 gene expression levels. Materials and methods from May 2016 to September to the first Zhengzhou University Department of Hematology Affiliated Hospital of hospitalization and consider AL patients and has been diagnosed as AL to hospital intensive treatment patients and healthy volunteers 2ml bone marrow specimens, specimens were collected for each number, the name of the patient, hospital number, date and the number of the corresponding samples. Bone marrow mononuclear cells were isolated by lymphocyte separation medium, Trizol was used to extract bone marrow mononuclear cells total RNA, agarose electrophoresis of RNA integrity, measured the purity and concentration of RNA UV spectrophotometry, abandon RNA unqualified (RNA is not complete, or not 娴撳害杩囦綆)鐨勬爣鏈，

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