【作者】 杨琰；

【导师】 黄士昂；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 第一部分C-KIT/D816V突变在急性髓系性白血病中的发生率调查目的：分析C-KIT/D816V突变在中国人AML患者中的发生率。方法：收集2008年至2011年送至干细胞中心行C-KIT/D816V检测的AML外周血标本286例,提取者外周血单个核细胞,采用Real-time Quantitative PCR SYBR green染料法检测C-KIT D816V突变。结果：286例AML患者中,有8例(2.8%,其中6例男性,2例女性)患者发生了C-KIT/D816V突变,其在男性患者和女性患者中的发生率分别为3.9%和1.5%,在男性患者中的发生率高于女性,差异有统计学意义(P<0.05)。8例C-KIT突变患者中有5例M2,1例M4,另外2例患者FAB亚型诊断未明。在AML各亚型中的发生率分别为：M26.8%,M44.3%。结论：C-KIT/D816V突变最常发生在M2中,其次为M4。在男性患者中的发生率高于女性。本研究从流行病学的角度为C-KIT/D816V突变在中国人AML及其亚型中的发生率提供了重要依据。第二部分关于携带C-KIT/D816V突变及C-KIT/WT的急性髓系白血病PP2A蛋白活性变化的研究目的：从流行病学的角度探讨携带C-KIT/D816V[位于C-KIT激酶区(C-KIT kinase domain, C-KIT/TKD)]突变的急性髓细胞性白血病患者及携带野生型C-KIT (C-KIT/WT)的急性髓细胞性白血病患者单个核细胞中蛋白磷酸酯酶2A(protein phosphotase 2A, PP2A)的活性变化,并对携带C-KIT/N822K(位于C-KIT/TKD)突变的AML细胞株Kasumi-1及携带野生型C-KIT的AML细胞株HL60中PP2A活性进行了比较。方法：选取8例C-KIT/D816V突变的AML患者,12例C-KIT/WT AML患者及20例健康对照,提取单个核细胞,使用PP2A免疫沉淀磷酸酶检测试剂盒检测PP2A活性。体外培养HL60及Kasumi-1细胞株,使用PP2A免疫沉淀磷酸酶检测试剂盒检测PP2A活性。使用SPSS16.0对数据进行分析。结果：C-KIT/WT AML组患者单个核细胞PP2A的活性(565.69±122.56pmol)与正常人(706.02±154.22pmol)比较有所降低,而C-KIT/D816V AML组(419.01±96.97pmol)活性最低,各组之间差异有统计学意义(P<0.05)。Kasumi-1细胞PP2A活性(886.420±152.187 pmol)与HL60细胞(1196.442±219.483pmol)比较有所降低,差异有统计学意义(P<0.05)。结论：AML患者PP2A活性有所降低,且携带C-KIT/TKD的AML患者及细胞株中PP2A活性与携带C-KIT/WT的AML患者或细胞株比较有所降低,PP2A蛋白活性与AML的发生、发展密切相关,对AML的诊断及治疗可能有参考价值。第三部分FTY720对携带C-KIT激酶区突变的AML体外抑制作用的机制研究目的：本研究探讨FTY720对携带C-KIT/TKD突变的AML细胞系Kasumi-1及未携带C-KIT/TKD突变的AML细胞系HL-60细胞增殖、凋亡的影响，并对FTY720对两种细胞作用的进行了比较了,探讨其可能机制。方法：体外培养AML细胞株HL60及Kasumi-1,使用FTY720(2.5,5,10和20μM)处理HL-60、Kasumi-1细胞24小时,应用CCK8试剂盒检测细胞的增殖状况。使用FTY720(10μM)处理细胞36h,应用流式细胞术检测细胞的凋亡率。使用FTY720(5μM,10μM)处理细胞5h,应用PP2A免疫沉淀磷酸酶活性检测试剂盒测定细胞PP2A活性。用FTY720(5μM)处理细胞5h,应用Western Blot检测PP2A的表达水平。结果：FTY720能有效抑制HL60和Kasumi-1细胞的增殖,随药物作用浓度的增加,抑制作用越强,作用24h后FTY720对HL60和Kasumi-1的IC50分别为13.5μM和20μM,P<0.05,两者差异有统计学意义。FTY720作用于HL60和Kasumi-1后,两种细胞的凋亡率均增加,且Kasumi-1的凋亡率增加更显著。FTY720能增强HL60和Kasumi-1细胞的PP2A活性,但不改变PP2A的表达水平。PP2A抑制剂OA能抑制FTY720所致的PP2A活性增强,同时也能减轻FTY720所致的细胞毒性作用。FTY720不改变PP2A的表达水平。结论：FTY720能通过活化PP2A抑制HL60和Kasumi-1细胞的增殖并诱导两种细胞发生凋亡,且对后者的作用强于前者。FTY720对AML,尤其是难治性AML可能有治疗价值。更多还原

【Abstract】 PART I Detection of C-KIT/D816V mutation in Acute Myeloid Leukemia with Different Subtypes in ChinaObjective To investigate and analyze the incidence of C-KIT/D816V mutation in AML patients in China.Methods Blood samples from 286 AML patients with various French-American-British (FAB) classifications were collected from Center for Stem Cell Research and Application between 2008 and 2011. The diagnosis of AML and the assignment of FAB classification were based on morphology and immunophenotype. Then the quantitative realtime-PCR was used to examine the C-KIT/D816V mutation.Results Of the 286 AML samples studied,8 were shown to have a point mutation at D816V, including 6 male cases and 2 female cases. Its incidence in male and female was 3.9% and 1.5% respectively, thus it was more inclined to occur in male than female (P< 0.05). This mutation was typically located within the M2 (5 cases) and M4 (1 cases) subclasses, with the other 2 mutation AML cases not definitely diagnosed. In the present work, the D816V mutation occurred in 6.8% of M2,4.3% of M4 subclasses, and the overall incidence of C-KIT/D816V was 2.8% in AML.Conclution C-KIT/D816V most frequently occurs in M2 subutype, then M4 in AML, and it is more inclined to occur in male than female. Our research provided significant evidence for the incidence of C-KIT/D816V mutation in AML and its subtypes in China. partⅡAnalysis of Changes of the Activity of PP2A in Acute Myeloid LeukemiaObjective To analyze the changes of PP2A activity in AML patients with C-KIT/WT and C-KIT/D816V and compare the PP2A activity in HL60 cell harboring C-KIT/WT and kasumi-1 cell harboring C-KIT/TKD.Methods Eight AML patients harboring C-KIT/D816V mutation, twelve AML patients harboring wide-type C-KIT (C-KIT/WT) and twenty healthy controls were recruited and informed consent at Union hospital of Wuhan in this study. Real-time quantitative PCR was used to identify C-KIT/WT and C-KIT/D816V. Peripheral blood mononuclear cells (PBMCs) of patients with AML and normal controls were collected. In addition, HL60 and kasumi-1 cells were cultivated in vitro. The activity of PP2A in the PBMCs of patients and the cell lines was assayed with a PP2A Immunoprecipitation Phosphatase Assay Kit, the data was analyzed by SPSS 16.0.Results We detected PP2A activity of all samples with PP2A Immunoprecipitation Phosphatase Assay Kit. The results indicated that the PP2A activity in AML patients harboring C-KIT/WT(565.69±122.56 pmol) was obviously lower than the normal controls(706.02±154.22 pmol), and the PP2A activity in AML patients harboring C-KIT/D816V(419.01±96.97 pmol) was the lowest. There was significant difference between the three groups (P<0.05). In addition, PP2A activity in Kasumi-1 cell(886.420±152.187 pmol) was obviously lower than HL60 cell(1196.442±219.483 pmol). There was significant difference between them (P<0.05).Conclution It is concluded that from the standpoint of prevalence, PP2A activity is decreased in patients with C-KIT/TKD AML. In addition, compared with AML cells lines harboring C-KIT/WT, PP2A activity of AML cells harboring C-KIT/TKD is obviously lower. PP2A protein plays a key role in the genesis and development of AML, and may imply a diagnosis and treatment of AML, especially the refractory C-KIT/TKD AML. PARTⅢCytotoxic Effect of FTY720 on AML Cell Lines harboring C-KIT/TKD and C-KIT/WT and Its MechanismObjective To observe the cytotoxicity of FTY720 in C-KIT/TKD AML and develop FTY720 as a novel therapy for C-KIT/TKD AMLMethods Cell lines were treated with FTY720 alone or combined with specific PP2A inhibitor okadaic acid (OA) in culture. Cell growth was assessed using the CCK8 Kit. Cell apoptosis was analyzed by flow cytometry after dual staining with annexinⅤ/ propidium iodide. PP2A activity was assayed with a PP2A Immunoprecipitation Phosphatase Assay Kit. Western blotting was used to analyze the protein changes. All experiments were repeated three times. Statistical analysis was performed using SPSS 16.0.Results FTY720 induced toxicity in all AML cells, including Kasumi-1 and HL60 in a time-and dose-dependent manner. PP2A inhibitor OA rescued these cells from FTY720-induced apoptosis. Furthermore, Increased PP2A activity was detected after FTY720 treatment. When cells were pretreated with OA, PP2A activity was partially rescued. Finally, PP2A expression remained unchanged after FTY720 treatment. Our results strongly suggested that FTY720-induced cytotoxicity was mediated by PP2A activation without altering its expression. Interestingly, it was observed that cells harboring C-KIT/TKD mutation were more sensitive to this agentConclusions Our study provides evidence for the PP2A-dependent toxicity of FTY720 in AML cells, and these effects of FTY720 appear to be most marked in C-KIT/TKD AML where conventional chemotherapeutic approaches are most likely to fail. The significant preclinical efficacy of FTY720 indicates that it may be valuable therapeutic agents for refractory C-KIT/TKD AML.更多还原