【作者】 赵宸；

【导师】 司维柯；

【作者基本信息】 第三军医大学 ， 临床检验诊断学， 2010， 硕士

【摘要】 目的：Wnt蛋白家族包括多个成员且序列和信号传导方式高度保守,在调控胚胎发育中起重要作用,Wnt5a是其家族成员之一,该基因在各类肿瘤中的作用是近年关注的热点之一。与Wnt5a相关的研究涉及多种肿瘤,并且在不同肿瘤中的表现不一。髓系白血病在中国的发病率持续升高,且国内外罕见Wnt5a与髓系白血病关系的报道,我们前期研究已证明Wnt5a具有抑制白血病细胞株增殖的作用,其表达缺失可能与白血病发生有关,但明确的作用机制尚未深入研究。本研究拟观察髓系白血病病人治疗缓解前后骨髓有核细胞Wnt5a启动子甲基化水平的变化和去甲基化药物对白血病细胞Wnt5a启动子甲基化水平的影响,进一步探讨它表达下调或沉默的机理以及与髓系白血病发生的关系,从而为白血病提供新的肿瘤诊断标志物和基因治疗靶点。方法：1.MSP法检测30例急性髓细胞白血病初诊病例和31例急性髓细胞白血病缓解病例骨髓有核细胞Wnt5a启动子甲基化水平。2.MSP法检测17例慢性粒细胞白血病慢性期病例标本和3例急变期病例标本Wnt5a启动子甲基化水平。3.淋巴细胞分离液和细胞培养技术分离培养髓系白血病原代细胞和骨髓基质细胞,并应用RT-PCR方法检测2例培养的白血病原代细胞和骨髓基质细胞中Wnt5a的表达。4.MSP法检测白血病原代细胞和骨髓基质细胞中Wnt5a启动子甲基化情况。5.MTT法检测不同作用时间点和不同浓度去甲基化试剂Adc处理条件下的各组U937细胞的增殖情况,并确定后期实验的最适药物用量和作用时间。6.去甲基化试剂Adc处理U937白血病细胞株,检测不同药物浓度作用下Wnt5a的表达水平和Wnt5a基因启动子甲基化的水平的差异。7.流式细胞术检测去甲基化试剂Adc处理U937细胞2d后细胞周期的变化情况。结果：1. Wnt5a启动子甲基化情况：30例急性髓细胞白血病初诊病例中22例出现甲基化现象,启动子甲基化率为73.3%(22/30)；31例急性髓细胞白血病缓解病例中3例出现甲基化现象,甲基化率为9.7%(3/31)2.17例慢性粒细胞白血病慢性期病例标本和3例急变期病例标本中,Wnt5a启动子都未见甲基化。3.分离获得悬浮生长的白血病原代细胞和贴壁生长的骨髓基质细胞中,骨髓基质细胞Wnt5a表达阳性,而白血病原代细胞中未检测到Wnt5a表达。4.骨髓基质细胞中Wnt5a启动子没有发生甲基化,而在白血病原代细胞中分别表现为部分甲基化和完全甲基化两种情况。5.不同浓度去甲基化试剂Adc处理U937细胞不同时间,最终确定适用于U937细胞的Adc作用时间为48h,浓度范围为3μmol/L～10μmol/L。6.Adc处理后U937细胞Wnt5a启动子甲基化水平下调,mRNA表达恢复,且在一定浓度范围内,Wnt5a的表达量与Adc药物浓度有关。7.与对照组细胞相比,Adc处理后U937细胞G2期增高,表明Adc处理后,细胞周期被阻滞在G2期。结论：1.Wnt5a启动子甲基化现象在急性髓细胞白血病初诊病例中阳性率为73.3%,明显高于缓解病例阳性率9.7%,证实了我们前期研究发现白血病标本中普遍存在Wnt5a表达缺失是因其启动子异常甲基化所致。2.治疗缓解后Wnt5a启动子甲基化状态明显下降,表明我们前期研究发现治疗缓解病例Wnt5a表达恢复的原因是经治疗去除了其启动子的甲基化状态。研究结果提示Wnt5a启动子的异常甲基化可能与白血病细胞的恶性增殖有关,Wnt5a表达水平变化和启动子甲基化状态可能成为判断病情发展和预后的实验室诊断新指标。3.慢性期和急变期的慢粒病人骨髓有核细胞Wnt5a启动子均未见甲基化现象。前期研究中Wnt5a在慢粒病人和K562细胞中不表达或低表达,在mRNA水平上与在急性髓系白血病中极为类似；但启动子甲基化研究提示Wnt5a在慢粒病人中表达下调原因不同于急性髓细胞白血病,可能受其他机制调节,有待进一步研究。4.白血病病人的骨髓基质细胞中Wnt5a高表达,且启动子未见甲基化,而白血病肿瘤细胞Wnt5a低表达且启动子发生明显甲基化,说明白血病病人骨髓中的基质细胞可能未发生异常,Wnt5a启动子甲基化可能特异的出现在白血病细胞等恶性细胞中,是急性髓细胞白血病中Wnt5a低表达的主要原因。5.去甲基化试剂Adc作用后,白血病细胞U937增殖能力减低,提示Wnt5a的表达可能抑制白血病细胞增殖。6.Adc处理后,Wnt5a表达恢复,U937细胞G2期增高,提示Wnt5a可能将细胞周期阻滞在G2期,细胞不能进行有丝分裂,从而抑制了细胞的增殖,但去甲基化试剂不能完全改变U937细胞Wnt5a启动子异常甲基化情况,说明仍有其他机制维持白血病细胞的异常甲基化,其具体机制仍需进一步研究。以上研究提示,Wnt5a启动子甲基化水平在急性髓细胞白血病病例中普遍升高,而在治疗缓解病例中降低,印证了前期研究,说明急性髓细胞白血病中Wnt5a启动子甲基化是导致Wnt5a表达缺失的分子机制,而在慢性粒细胞白血病各病程中Wnt5a的表达缺失不由类似的调控因素控制。本组实验与前期研究共同证明Wnt5a在白血病病例中发挥抑癌基因的作用,其表达缺失是白血病发生的机制之一,并受到表观遗传学方法的调节,其中存在“Wnt5a启动子异常甲基化-Wnt5a表达下调-白血病发生”的逻辑联系。更多还原

【Abstract】 ObjectiveWnt family contains several members and shows a highly regulated pattern of expression and plays distinct roles during development and tissue homoeostasis. Wnt5a, a member of the Wnt family, has been recently found to be correlated with tumorigenesis. The effects of Wnt-5a in kinds of tumors are significantly different. The incidence of AML maintains a pretty high level in China. There are infrequent reports about the relationship between Wnt5a and AML at home and abroad. Our earlier studies of the gene demostrate that the low expression of Wnt5a is universal in leukemia, and the expression of Wnt5a was recovered in the patient with leukemia-CR. Wnt5a may play a role of anti-oncogene in leukemia, and the loss expression of Wnt5a is one of the mechanisms of leukemia genesis. However, the mechanisms of expression silence are not clear. This study was aimed to detect the methylation status of Wnt5a promoter in the acute myeloblastic leukemia patients’and CR patients’bone marrow, and take research on the relationship between aberrant methylation status of Wnt5a gene promoter and mechanism of myeloblastic leukemia, in order to find a new tumor marker and a potential therapeutic target for AML.Methods1. The methylation status of Wnt5a promoter was analyzed by methylation-specific PCR(MSP) and verified in both 30 AML patients and 31 AML-CR patients.2. The methylation status of Wnt5a promoter was analyzed by MSP and verified in 17 CML patients in chronic phase and 3 CML patients in blast crisis.3. The expression of Wnt5a gene was detected with RT-PCR in primary AML cells and marrow stromal cells.4. The methylation status of Wnt5a promoter in primary AML cells and marrow stromal cells was analyzed by MSP. 5. MTT assays were performed to detect the endogenous Wnt5a effect on the proliferation of U937 and the proper treatment time and dosage.6. RT-PCR was performed to detect the expression of Wnt5a while MSP was performed to detect the status of methylation of Wnt5a gene promoter before and after treating with Adc.7. Flow cytometry was performed to assay the cell cycle of U937 cells after being exposed to Adc for 2d.Results1. Methylation of Wnt5a gene promoter were found in bone marrow of samples from 30 AML patients at 73.3% by the methylation-specific PCR; methylation of Wnt5a gene promoter were found in 31 AML-CR patients at 9.7% by the methylation-specific PCR.2. Methylation of Wnt5a gene promoter were not found in bone marrow of samples from either 17 CML-CP patients or 3 CML-BC patients.3. The bone marrow stromal cells and primary AML cells were isolated by gradient centrifugation and purified by anchoring culture. Wnt5a mRNA were expressed in stromal cells but not in AML cells analysed by RT-PCR.4. Methylation of Wnt5a gene promoter were not found in bone marrow stromal cells, and methylation status were partly found in AML cells.5. The modest concentration to treat U937 cells with Adc were 3μmol/L～5μmol/L for 48 or 72 hours separately and the abilities of proliferation were measured by MTT test.6. The Adc up-regulates the expression of Wnt5a mRNA and demathylated status of Wnt5a gene promoter after treating with modest range. And there was a linear relationship between expression levels and Adc concentration over a certain range.7. The number of U937 cells exposed to Adc at G2 phase was higher than the control.Conclusion:1. MSP analysis showed that CpG islands of Wnt5a promoter is hypermethylated in AML patients in acute phase(73.3%), which is rather higher than that in AML-CR patients(9.7%). Our previous study showed loss of Wnt5a expression may be related to the tumorigenesis of leukemia. This study is identical to this view at epigenetics level. It shows that hypermethylation of CpG islands of Wnt5a promoter induces the decline of Wnt5a expression and results in AML. 2. The positive frequency of methylation of Wnt5a promoter is lower after clinical treatment. It implied that aberrant DNA methylation of Wnt5a promoter may be related to the malign hyperplasia of leukemia cells in bone marrow. This could be used as a clinical index for appraising the therapeutic efficacy and prognosis.3. The aberrant DNA methylation of Wnt5a promoter were not found in either 17 CML-CP patients or 3 CML-BC patients. We have found that the Wnt5a mRNA expression decreased in CML patients and K562 cells, which is similar to that in AML. However, the contrary results in methylation study suggest a different regulating mechanism which calls for further research.4. We initially established the methods of bone marrow stromal cell and primary leukemia cell culture. Wnt5a was found high expression in mRNA level but unmethylation in epigenetics in bone marrow stromal cells of AML patients. It suggests the aberrant DNA methylation of Wnt5a promoter specifically associated with the tumor cells.5. After being exposed to demethylation reagent Adc, the demethylation of Wnt5a promoter were found in leukemia cell lines U937, and the expression of Wnt5a was increased. Compared with the control, the growth of leukemia cell line U937 exposed to demethylation reagent was significantly inhibited, and the number of cells in G2 phase rised. It is implicated that Acd can block cell cycle of U937 cells in G2 phase and inhibit cell proliferation.Our studies demostrate that:the positive frequency of the methylation of Wnt5a promoter increased in AML acute phase and decreased in AML-CR phase.The molecular mechanisms of Wnt5a gene silencing in AML is the aberrant DNA methylation of Wnt5a promoter. This rule is not applicable to CML. It is proved that Wnt5a plays a anti-oncogene role in leukemia and the loss of expression of Wnt5a was(is) one of the mechanisms of leukemia genesis in our experiments and previous studies. There is a relation among the aberrant methylation of Wnt5a promoter, the low expression of mRNA and the pathogenesis of acute myeloid leukemia.更多还原