【作者】 朱亚楠；

【导师】 姜中兴；

【作者基本信息】 郑州大学 ， 血液内科， 2012， 硕士

【摘要】 背景与目的急性白血病(Acute Leukemia, AL),已被认为是严重威胁人类身体健康的恶性肿瘤之一。该疾病的特征为原始和(或)幼稚细胞增殖发生异常,同时分化、凋亡也会受到不同程度抑制。AL的发病机制目前尚未十分明确,但有研究显示它的发生发展与原癌基因的活化、抑癌基因的灭活有关。抑癌基因是启动细胞癌变的主要因素,对细胞生长起负调控作用,基因缺失、突变或错位等诸多因素均可引起抑癌基因功能异常。生长抑制因子-1(Inhibitorof growthl,ING1)是新近发现的一种抑癌基因,在几乎所有的正常细胞中较广泛表达。ING1基因通过不同的转录剪切方式翻译出不同分子量的蛋白质,p33INGlb是ING1编码的重要蛋白质,在肿瘤的发生发展中起着非常重要的作用,其磷酸化状态决定它参与细胞周期调控等多种生物学功能。在正常组织细胞中,p33INGlb蛋白在GO进入G1期时表达量减少,在G1晚期又出现增加趋势,在S期达到最大值,接着在G2期表达水平降低。有研究表明,ING1基因核表达减少的现象在一部分良性肿瘤以及几乎所有恶性肿瘤的组织细胞中均可出现。目前p33INGlb在急性白血病患者中的表达及其可能参与的发病机制已有报道,但是应用逆转录聚合酶链反应(reverse transcriptase-polymerase chain reaction)RT-PCR法检测不同类型急性白血病患者p33INGlb的表达情况以及其与AL骨髓外浸润及复发的相关报道较少。本实验通过RT-PCR法检测p33ING1b在不同类型和不同疾病阶段急性白血病患者的骨髓单个核细胞中表达情况及表达水平的差异,分析p33INGlb与AL患者临床表现的相关性,进而探讨其在AL发生发展中可能参与的作用机制。材料与方法1研究对象65例病例来自2010年12月-2012年3月我院住院或门诊急性白血病患者,平均年龄34(15-68)岁。(1)初治组46例；男性20例,女性26例,其中急性髓系白血病(AML)32例(AML-M219例,AML-M37例,AML-M52例),急性淋巴细胞白血病(ALL)14例；(2)完全缓解组12例；AML8例,ALL4例；(3)复发组7例；男3例,女4例；(4)正常对照组；为8名骨髓象正常的非恶性血液病患者。2实验方法RT-PCR法检测p33ING1bmRNA的表达水平：取骨髓标本,分离单个核细胞后Trizol法提取实验细胞总RNA并检测其质量及浓度,逆转录成cDNA, PCR扩增,琼脂糖凝胶电泳后,将琼脂糖凝胶放入凝胶成像扫描仪中紫外线下成像,用凝胶图像分析系统观察、分析实验结果并拍照。3统计分析实验结果应用SPSS17.0统计学软件分析,对定量资料使用平均值±标准差即x±sd,采用t检验对两样本均数进行比较,应用方差分析对多样本均数比较,定性资料采用卡方检验及Fisher’s精确概率试验,采用Spearman分析相关性,以a=0.05为检验水准。结果1. p33ING1bmRNA在AL初治组、复发组、缓解组中的阳性率分别为45.7%(21/46),42.9%(3/7),58.3%(7/12), p33ING1bmRNA的表达水平分别为0.203±0.106,0.389±0.027,0.627±0.202；正常对照组的阳性率为87.5%(7/8),它们的阳性细胞率及表达水平明显低于对照组。复发组的阳性率及表达水平与缓解组比较,差异具有统计学意义(p<0.05)；复发组与初治组的阳性率差异无统计学意义(p>0.05),但表达水平差异有统计学意义(p<0.05)；初治组的表达水平及阳性率分别为0.203-0.106、45.7%与缓解组的阳性率58.3%及表达水平0.627±0.202比较差异有统计学意义(p<0.05)2. AML患者p33ING1b的阳性表达率为45.5%(20/44)而ALL为52.38%(11/21),两组平均表达水平分别为0.491±0.173、0.565±0.221,阳性表达率及平均表达水平差异无统计学意义(p>0.05)。3.p33ING1bmRNA的表达水平与AL的骨髓外浸润、患者的性别、年龄无明显相关性(p>0.05)结论1.p33ING1bmRNA在急性白血病中低表达,在缓解组的表达水平较初治组和复发组高,差异有统计学意义,提示p33ING1b的低表达可能与急性白血病的发生发展有关。2.p33ING1bmRNA在初治ALL和AML患者中均低表达,差异无统计学意义,提示其低表达与白血病的类型可能无关。3.p33ING1bmRNA的表达水平与急性白血病患者的性别、年龄、髓外浸润等临床特征无相关性。更多还原

【Abstract】 Background and PurposeAcute Leukemia (Acute Leukemia, AL), it is a serious threat to human health of cancer for it’s increased high morbidity and mortality. The characteristics of AL is the original or childish abnormal cell proliferation, differentiation and apoptosis is inhabited. The pathogenesis of AL is not clear, but research shows that it has contact with protocarcinogenic gene activation and the inactivation of tumor-suppressor gene. Tumor-suppressor gene is a important change factor that leads to cell abnormal.This abnormal function of cells is caused by many reasons such as gene loss, mutation, displacement and so on.It play a negative role in cell growth. Tumor suppressor gene is an initiation factor for malignant cells,which caused by many reasons causing dysfunction such as gene deletion, mutation, dislocation, and has the negative regulation of cell growth. Growth inhibitory factor-1(Inhibitor of growthl, ING1) is a recently discovered tumor suppressor gene, widely expressed in normal human tissues and a variety of tumor tissue, and related to cell cycle regulation, through different transcription cut way to translate the different molecular proteins, P33ING1b is the main product of its transcription and translation as well as important tumor suppressor gene. The phenomenon of its nuclear expression decreased and cytoplasmic over-expression may occur in some benign and almost all malignant tissue cells. ING1gene translate a molecular weight of alien protein by different shear mode and p33ING1b is the protein encoded by ING1,involved in a variety of biological function mechanism such as cell cycle regulation depends on its phosphorylation state, playing a very important role in tumor development. In normal tissue cells, p33ING1b protein reduced from GO to G1phase, increased in the G1late phase and reached its maximum in the S phase, and then lower in the G2phase.In recent years, related studies have shown that P33INGlb protein expressed in the nucleus of bone marrow mononuclear cells in normal human and acute leukemia patient. The possible mechanisms of p33ING1b in the pathogenesis of acute leukemia have been reported, but the detection of p33ING1b by PCR in acute leukemia patients has not been reported in the domestic. This issue through real-time quantitative PCR to found the difference of the INGlb gene expression level between normal human and AL patients with different stages, analysing the relatation between p33ING1b and the clinical relevance of AL patients and exploring its possible mechanism in the AL.Methods1. An object of study:65cases from our hospital or clinic patients with acute leukemia from December2010to March,2012, the average age is34(15-68) years.(1) The untreated group,46cases;20males and26females, including32cases of acute myeloid leukemia (AML)(AML-M219cases,7cases of AML-M3AML-M52cases),14cases of acute lymphoblastic leukemia (ALL);(2) complete remission group,12cases;8cases of AML,4cases of ALL;(3) The group of7cases of recurrence;3males and4females;(4) normal control group;8cases non-hematologic malignancies patients with normal bone marrow.2. RNA extraction and reverse transcription:bone marrow samples, separate mononuclear cells,then extract experiment total cellular RNA by Trizol and detect the quality and concentration,maging under ultraviolet light, and then systematic observation, analysis the experimental results and photographed using gel image analysis. 3. Statistical analysis:Using Software SPSS17.0analyze the data. Quantitative data were expressed as mean±tandard deviation (x±SD); Paired means were analyzed by Independent-Samples t-test, while Various means were analyzed by variance analysis. Qualitative data were analyzed by chi-square test; The standard of significant level was a=0.05.Result1. p33ING1b in the untreated group of AL, the positive rate of45.7%(21/46), recurrent group was42.9%(3/7), the remission group was58.3%(7/12), the expression level of the P33were0.203±0.106,0.389±0.027,0.627±0.202; normal control group, the positive rate was87.5%(7/8). The positive rate of the recurrence group was significantly lower than the positive rate of the remission group and normal control group, the difference between the recurrence group and the latter two groups was statistically significant (p<0.05); the difference of the positive rate between the early treatment group and remission group had no statistically significant (p>0.05) but the difference of expression level was statistically significant (p<0.05); the differences of expression levels and the positive rate between the recurrence group and the remission group were statistically significant (p<0.05);2. the positive expression rate was45.5%(20/44) in AML,52.38%(11/21) in ALL, the expression levels of two sets were0.491±0.173,0.495±0.221, the differences of the positive rate and expression levels had no statistically significant (p>0.05).3. The expression level of p33ING1b mRNA has no significant correlation with the bone marrow and infiltration, patients for gender, age,(p>0.05).Conclusion1. p33ING1b low expressed in the AL; the expression of recurrence is lowest, the difference had statistics meaning, this may indicate lower expression of p33ING1b gene may play a role in the occurrence and development of acute leukemia. 2. p33ING1b has low expression level in the AML group and ALL group group, the difference was statistically significant, suggesting that the low-expression of p33ING1b may had no meaning to the type of acute leukemia.3. The expression level of p33ING1b in acute leukemic patients has no relevation witn gender, age, and infiltration.更多还原