本文選題：Aurora基因　切入點：急性白血病　出處：《鄭州大學》2017年碩士論文　論文類型：學位論文

【摘要】：Aurora家族基因包括Aurora-A、B、C,它們表達的蛋白是一組高度相關(guān)的Aurora激酶家族,在正常細胞中對調(diào)控細胞分裂起著至關(guān)重要的作用,Aurora-A激酶的主要功能是中心體的調(diào)節(jié)和有絲分裂紡錘體的形成,Aurora-B激酶是一種旅客蛋白,其功能主要是確保染色體的分離和胞質(zhì)分裂,Aurora-C激酶主要在減數(shù)分裂中起重要作用,也有研究顯示Aurora-C激酶也是一種旅客蛋白,對Aurora-B激酶的功能起補充作用,Aurora激酶的失調(diào)或異常會引起細胞分裂異常,進而促使染色體不穩(wěn)定和腫瘤的形成。從目前研究來看,Aurora-C激酶主要參與生精過程,對有絲分裂特別是對中心體的功能我們了解的還不夠深入,尤其是其對腫瘤發(fā)生、發(fā)展、耐藥的機制及意義,有研究認為其對腫瘤不起作用,有研究認為其促進Aurora-B激酶的功能,有研究認為Aurora-C激酶也能促進細胞轉(zhuǎn)化和腫瘤形成。在多種腫瘤中Aurora-A和B激酶高表達,其表達異常能夠激活調(diào)節(jié)細胞增殖的相關(guān)信號傳導通路,阻礙細胞分化與凋亡,其表達水平和腫瘤的分期、預后等都有一定的相關(guān)性,然而,Aurora基因與急性白血病(AL)臨床指標的關(guān)系鮮有研究,所以Aurora-A、B、C mRNA表達水平與急性白血病類型、初治白血病細胞(WBC)水平、乳酸脫氫酶(LDH)水平、危險分層及預后不良表面抗原(CD34、CD56、CD71)等臨床指標的關(guān)系將在本實驗中要進一步研究。研究目的本研究中初步采用熒光定量PCR對初診和化療完全緩解的AL病人與健康志愿者骨髓單個核細胞中AUR-A、B、C mRNA表達水平進行檢測,并探討其與AL臨床指標的關(guān)系及3基因表達水平之間的相關(guān)性。材料與方法收集2016年5月至9月至鄭州大學第一附屬醫(yī)院血液科住院并考慮AL患者和已明確診斷為AL來院強化治療患者以及健康志愿者的骨髓標本2ml,每個標本進行編號,搜集患者姓名、住院號、標本日期與編號相對應。應用淋巴細胞分離液分離骨髓單個核細胞,應用Trizol提取骨髓單個核細胞總RNA,瓊脂糖電泳鑒定RNA完整性,紫外分光光度計檢測RNA純度和濃度,舍棄RNA質(zhì)量不合格(RNA不完整,不純或者濃度過低)的標本,質(zhì)量合格標本加入RNA酶抑制劑并應用無RNA酶水調(diào)整RNA濃度至400ng/μL,然后暫時存放到-80℃冰箱保存。初診未明確診斷患者明確診斷后剔除非急性白血病標本,收集剩余標本的臨床資料如性別、年齡、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淋巴細胞白血病)、初診WBC計數(shù)、幼稚細胞比例、初診LDH水平、染色體類型(正常核型、t(15;17)、t(8;21)、t(9;22)、復雜核型、其它)、危險分層(低危、中危、高危)、化療緩解情況(1療程緩解與否)、基因突變情況和與預后相關(guān)的細胞CD分子陽性百分比(CD34、CD56、CD71),剔除臨床資料不完整的標本,符合條件的標本應用熒光定量PCR檢測Aurora-A、B、C mRNA相對表達量,其等于2-ΔCT,其中ΔCT=目的基因CT-內(nèi)參基因CT,內(nèi)參基因選用GAPDH。應用SPSS 17.0軟件對數(shù)據(jù)進行統(tǒng)計分析,對2組間表達差異采用非參數(shù)檢驗2個獨立樣本檢驗,對于多組間表達差異采用非參數(shù)檢驗k個獨立樣本檢驗,組間差異有統(tǒng)計學意義者采用Bonferroni法進行多重比較,P0.05表示差異有統(tǒng)計學意義,對于定量資料的相關(guān)性分析采用Spearman相關(guān)性分析。結(jié)果1 Aurora家族基因在不同分組中的表達差異Aurora-A、B、C基因mRNA表達水平在73例初診AL患者均高于14名健康志愿者和20例化療完全緩解AL患者(P0.01),而3種基因mRNA表達水平在健康組和緩解組差異均無統(tǒng)計學意義(P0.05),在急性淋巴細胞白血病(ALL)組均高于急性髓系白血病(AML)組(P0.01)。Aurora-A、B、C基因mRNA表達在CD34、CD71、CD56陰性與陽性組差異無統(tǒng)計學意義(P0.05),3種基因表達在不同性別組和1療程化療緩解與未緩解組差異均無統(tǒng)計學意義(P0.05)。Aurora-A、B、C表達在不同染色體分組中差異無統(tǒng)計學意義(P0.05)。AUR-A、B、C mRNA表達在高危組均高于低危組(P0.05),在中危組和低危組與高危組和中危組Aurora-A、B、C表達差異均無統(tǒng)計學意義(P0.05)。2 Aurora家族基因與臨床指標的相關(guān)性73例初診AL患者中,Aurora-A mRNA表達水平與初診LDH水平和危險分層呈顯著正相關(guān)(r=0.279,P=0.017;r=0.314,P=0.007);Aurora-B與初診LDH水平和危險分層呈顯著正相關(guān)(r=0.277,P=0.018;r=0.349,P=0.002),Aurora-A、B均與年齡、初診WBC水平、初診骨髓幼稚細胞比例和1療程是否緩解無顯著相關(guān)性;Aurora-C表達水平與初診WBC水平、初診LDH水平和危險分層呈顯著正相關(guān)(r=0.263,P=0.025;r=0.348,P=0.003;r=0.376,P=0.001),與年齡在0.05水平顯著負相關(guān)(r=-0.241,P=0.040),而與初診骨髓中幼稚細胞比例和1療程是否緩解無顯著相關(guān)性。3 Aurora家族基因之間表達水平的相關(guān)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表達水平均呈顯著正相關(guān)。結(jié)論1.Aurora-A、B、C mRNA在初診AL中相對于健康人和化療緩解患者高表達,且在ALL中高于AML,在T-ALL高于B-ALL,提示,Aurora家族基因可能均參與了AL的發(fā)生發(fā)展,并可以作為AL預后的標志物。2.Aurora-A、B、C mRNA表達水平與初診WBC水平、LDH水平和危險分層顯著正相關(guān),提示Aurora家族基因均可以作為危險分層或預后的指標。3.Aurora-A、B、C mRNA表達水平在AL中顯著正相關(guān),提示3基因在AL的發(fā)生、發(fā)展過程中可能存在功能互補或重疊。
[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 嫻撳害榪囦綆)鐨勬爣鏈，

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