【作者】 张利；

【导师】 张欣欣； 王军；

【作者基本信息】 郑州大学 ， 儿内科， 2003， 硕士

【摘要】 白血病是儿科常见恶性肿瘤，约占儿科恶性肿瘤的33％。其中急性淋巴细胞白血病(acute lymphoblastic leukemia，ALL)约占75％，急性髓细胞性白血病(acute myeloblastic leukemia，AML)约占20％。多数学者认为儿童白血病可能是遗传因素和环境因素共同作用的结果。而遗传性代谢酶纯合子缺失导致的机体对致畸变物质代谢解毒能力下降，可能是儿童白血病发生的遗传学基础之一。 作为人体Ⅱ相代谢酶，谷胱甘肽S-转移酶超家族(glutathione S-transferase superfamily，GSTs)具有多种生理功能。GSTs主要通过催化还原型谷胱甘肽(reduced glutathione，GSH)的巯基(-SH)与致畸变物质的亲电子核团结合，参与其代谢解毒过程。偶尔，在代谢过程中活化某些致畸变物质。此外，GSTs尚籍其谷胱甘肽过氧化物酶作用和氢过氧化物酶作用，催化还原型GSH与过氧化物和氢过氧化物结合，保护细胞免受氧化损伤。目前，人类的GSTs至少可以分为8类：alpha(α)、mu(μ)、theta(θ)、kappa(κ)、pi(π)、sigma(σ)、zeta(ζ)和omega(Ω)。其中，GSTM1基因(编码GSTμ)、GSTT1基因(编码GSTθ)在人群中呈多态性分布，已经成为肿瘤病因学研究的新热点之一。据报道，GSTM1、GSTT1基因多态性与多种恶性肿瘤的发生密切相关。但GSTM1、GSTT1基因多态性与儿童急性白血病发生的关系尚不清楚，国内未见此方面的报道。 此外，GSTs还参与白血病患者的多药耐药过程。GSTs的代谢底物广泛，尚包括部分抗肿瘤药物。GSTM1、GSTT1基因纯合子缺失的白血病患者对这些抗肿瘤药物的代谢能力下降，细胞内的药物浓度增加，可能影响患者的预后。但是，目前国内尚缺乏这一方面的研究报告。郑州大学2003届硕士学位论文谷肤甘肤S一转移酶MI、Tl基因多态性与儿童白血病的相关研究 鉴于此，本研究将通过提取全血基因组DNA、进行多重聚合酶链反应 (multiplex polymerase ehain reaCtlon，multi一pCR)的方法，检测GSTMI、GSTTI纯合子缺失基因型在急性白血病儿童和健康人群中的表达，探讨GSTMI、GSTTI基因多态性在儿童急性白血病病因及预后中的作用，为高危人群的筛查和预后评估提供理论依据。材料和方法:1.分组和临床资料的收集: (l)健康对照组:随机抽取146例健康献血者外周血作为对照; (2)ALL组:共计67例。其中，42例患者在确诊后接受了正规治疗; (3)AML组:共计犯例。2.实验方法: (1)采用蛋白酶K、无水乙醇，从全血组织中提取基因组DNA; (2)应用紫外分光光度计，测定DNA的纯度和浓度; (3)基因组DNA的multi一PCR扩增; (4)扩增产物经2%琼脂糖凝胶电泳(澳乙咤染色)(8OV60而n)，判定其基 因型。3.统计学分析: 采用了检验和精确概率法，分析急性白血病儿童和健康对照组之间GSTM卜GsT’T1基因纯合子缺失率是否存在差异;采用秩和检验、精确概率法和了检验，比较GSTMI、GSTTI纯合子缺失基因型与急性白血病儿童的高危临床因素如发病年龄、性别、发病时的外周血白细胞数目、FAB形态学分型、早期治疗反应等之间的相关关系。以a=0.05作为显著性水准。同时以GSTMI、GSTTI纯合子缺失基因型为暴露因素，计算比值比(odds ratio，OR)和95%可信区间 (eo吐dence Interval，Cl)。结果:1.健康人群的GSTMI纯合子缺失率为52.74%，GSTTI纯合子缺失率为49.32%， GST Ml一Tl纯合子联合缺失率为24.66%;2.ALL患者的GSTMI、GST Ml一Tl纯合子缺失率显著高于对照组(76.12%对郑州大学2003届硕士学位论文谷胧甘肤S一转移酶Ml、Tl基因多态性与儿童白血病的相关研究 52.74%，OR=2.856，95%Cl=1.493一5.465，P=0.001;50.74%对24.66%， OR二3 .148，95%Cl=1.712一5.789，P=0.0001);3.AML患者的GSTMI纯合子缺失率显著高于对照组(71.88%对52.74%， OR二2 .290，95%Cl=0.992一5.285，P=0.048):4.ALL和AML患者的GSTTI纯合子缺失率与对照组之间差异无显著性(分别 有62.69%对49.32%，OR二1.621，95%Cl=0.899一2.920，P=0.107;59.38%对 49.32%，OR井1.502，95%Cl=0.691一3.265，P=0.303);5.GSTMI、GSTTI基因型与ALL和AML患者的性别、发病年龄、发病时的外周血白细胞数、无显著相关关系FAB(French一American一British，FAB)形态学分型之间均(均有P>0.05);6.GSTMI基因纯合子缺失的ALL患者，与有GSTMI功能性等位基因的ALL 患者相比，其早期治疗反应差异无显著性(O卜2.500，95%Cl=0.262一23.864， P二0.733);但GSTTI基因纯合子缺失的ALL患者，其发生早期治疗反应差 的风险低于有GSTTI功能性等位基因的ALL患者(OR=0.238， 95%Cl=0.060~0.949，P=0.080)。结论:1 .GSTMI纯合子缺失基因型可能是儿童急性白血病发生的危险基因型;2.GSTMI、GSTTI纯合子缺失基因型对儿童急性淋巴细胞性白血病的发生有协 同作用;3.GST’T1纯合子缺失基因型可能在儿童急性白血病的发生过程中不起关键作 用，但具有该基因型的急性淋巴细胞性白血病患者其早期治疗反应较好;4.GSMI、GSTTI?更多还原

【Abstract】 Leukemia is a common childhood cancer that constituting about 33% of all childhood cancers. Acute lymphoblastic leukemia (ALL) constitutes 75% of childhood leukemia and acute myeloblastic leukemia (AML) constitutes 20% of childhood leukemia. Some investigators suggest that childhood leukemia may be linked to inherited genetic susceptibility and environmental factors. The null genotypes for some inherited metabolic enzymes that cause less activity to the mutagenic substances may be one of the bases of childhood leukemia susceptibility.As the phase II metabolizing enzymes, the glutathione S-transferase superfamily (GSTs) enzymes have numerous functions. GSTs can catalyze the conjugation of the electrophilic binding sites of the mutagenic substances with reduced glutathione, and thereby plays a significant role in the inactivation and, occasionally, the activation of some xenobiotics. In addition, GSTs can catalyze the peroxides and hydroperoxides with reduced GSH via glutathione peroxidase and hydroperoxidase activities, then protect the cells from oxygenation. Until present, human’s GSTs have been classified at least 8 groups, such as alpha(α), mu(μ), theta(θ), kappa(κ), pi(π), sigma(σ), zeta(ζ) and omega(Ω). GSTM1(μ ), GSTT1(θ) exhibit genetic polymorphisms in population distribution. Now, much attention are paid to the relationship between GSTM1 , GSTT1 genetic polymophisms and etiology of the malignancies. It has been reported that GSTM1, GSTT1 genetic polymorphisms are associated with several malignancies. But the relationship between the GSTM1, GSTT1 geneticpolymorphisms and childhood leukemia remains unknown in China.In addition, GSTs may contribute to anticancer drug resistance. The metabolic substances of GSTs are numerous including some anticancer drugs. The patients with the null genotypes may reduce the metabolic activity and decrease the concentrations of the anticancer drugs in the cells. So, GSTs genetic polymorphisms may influence the outcomes of the childhood leukemia. But there are no related reports in our country.In the present study, the genomic DNA of healthy controls and children with acute leukemia are analyzed, through multiplex polymerase chain reaction (multi-PCR) to determine whether GSTM1 , GSTT1 null genotypes could affect the etiology and outcome of childhood leukemia. It will contribute to the selection of high-risk population and the prediction of treatment outcome.Meterials and methods:1 . Categorizations and clinical data collection:(1) the healthy control group: One hundred and forty six healthy blood donors are selected at random as the controls;(2) ALL group: sixty seven cases. Forty two patients of this group received standard chemotherapy after diagnosis;(3) AML group: thirty two cases.2. Methods:(1) Using proteinase K and ethanol, the genomic DNA were extracted from the perpheral whole blood;(2) The purities and concentrations of the genomic DNA were assayed by spectrophotometer;(3) The genomic DNA were amplified by multi-PCR;(4) The amplified products were visuslized by electrophoresis in 2% agarose gels containing ethidium bromide at 80V for 60 min to determine the genotypes.3. Statistical analysis:The chi-square test and Fisher exact test are used to determine the differences indistribution of the GSTM1 ,GSTT1 null genotypes between the cases and the controls. The chi-square test, Fisher exact test and Pilixon rank-sum test are used to determine the association between the GSTM1 ,GSTT1 null genotype and some high-risk factors for the childhood leukemia. For example, sex, age and white blood cell (WBC) count at diagnosis , French- American-British (FAB) subgroups. The significant level is the rate of less than 0.05. When the null genotype is considered as exposed factor, Odds Ratio (OR) and 95% confidence interval (CI) are also calculated.Results:1. The frequencies of the GSTM1, GSTT1 and GST M1-T1 null genotypes are 52.74%, 49.32% and 24.66% in the healthy controls, respectively;更多还原