【作者】 孙洁；

【导师】 黄河；

【作者基本信息】 浙江大学 ， 内科学， 2004， 博士

【摘要】 多种研究显示，白血病与端粒酶活性密切相关。在白血病细胞株和白血病病人的细胞中都发现有端粒长度不同程度的缩短伴有端粒酶活性增高。但是端粒酶活性增高的机理尚不清楚。已知端粒酶活性受多种途径调控，其中端粒结合蛋白是近年来发现的一个新的调控途径。自第一个端粒结合蛋白人端粒重复序列结合因子TRF1于1995年被发现以来，已陆续有近10个端粒结合蛋白被发现，它们分布在端粒及周围，参与对端粒三级结构的维持、有丝分裂和细胞周期调控、染色体损伤修复等多项重要细胞生命活动的调控。其中主要的4个端粒结合蛋白TRF1，Pinx1，tankyrase和tankyrase-2对端粒酶活性具有调控作用，TRF1和Pinx1是端粒酶活性的负向调控因子，而tankyrase和tankyrase-2是正向调控因子。端粒结合蛋白在急性白血病细胞中的表达及与端粒酶活性的关系尚不清楚。 在本项研究中以这两对端粒酶活性的正向和负向调控因子为研究对象，系统研究它们在急性白血病细胞中的表达及与端粒酶活性的关系。进一步通过分析TRF1在恶性血液病细胞株细胞中的突变，从基因组水平探讨TRF1表达下降的原因。再进一步在急性早幼粒细胞性白血病细胞株NB4细胞的分化模型中分析4个端粒结合蛋白的表达改变及与端粒酶活性的关系，深入探讨端粒结合蛋白对端粒酶活性的可能调控机理。端粒结合蛋白在急性白血病细胞中的表达和与端粒酶活性的关系未见系统研究报道，TRF1在白血病细胞中的突变的分析未见报道，端粒结合蛋白在ATRA诱导的NB4细胞分化过程中的表达和调控的研究也未见报道。 本研究分三部分。 第一部分：以荧光定量RT-PCR技术定量检测4个端粒结合蛋白在30例初发的急性白血病标本中的mRNA水平的表达，并分析与端粒酶逆转录酶hTERTmRNA表达的关系。 30例急性白血病均为初发病人，其中男18例，女12例；平均年龄32岁；急性非淋巴细胞性白血病(ANLL)17例，急性淋巴细胞性白血病(ALL)13例；初诊时外周血白细胞计数大于100×10~9／L的10例，小于100×10~9／L的20例；染色体检查为异常核型的14例，正常核型的16例。浙江大学博士学位论文 hTE盯在急性白血病细胞中的表达(0.00244，O一0.0320)明显高于正常人骨髓单个核细胞(2.79x 10一4，o一0.0078)(P<0.00一)。但在ANLL和ALL细胞中对比表达无明显差异(P=0.742)。TRFI在急性白血病细胞中的表达(0.0!71，0.0127一0.193)明显低于正常人骨髓单个核细胞 (0.0457，0.00839~0.262)(P=0.019)，其中在ANLL细胞中的表达(0.0126，0.0127~0.0546)明显低于正常人骨髓单个核细胞中的表达(P<0.001)，但在ALL细胞中的表达(0.0745，1.92x 106一0.193)与正常人骨髓单个核细胞比较无显著性差异(P=0.761)，ANLL细胞中的表达明显低于ALL细胞中的表达(P=0.001)。相关性分析显示TRFI和hTERT在急性白血病细胞和正常人骨髓单个核细胞中的表达无显著相关性(二一0.!73，P二0.207)。Pinxl在急性白血病细胞中的表达(0 .003 12，542 x10一4一0.024)明显高于正常人骨髓单个核细胞(7 .89x!o一4，o一0.00863)(P=0.002)。在ANLL和ALL细胞中的表达无显著性差异(p=0.869)。pinxl不IJ hTERT在急性白血病细胞和正常人骨髓单个核细胞中的表达呈正相关(r=o.2%，p=0 .028)。tallkyrase在急性白血病细胞中的表达(0.0209，O川06~0.0997)也明显高于正常人骨髓单个核细胞(0.00288，2.65 X 10一4一0.0288)(P<0.00一)，在ANLL和ALL细胞中的表达无显著性差异 (P=0 .363)。tankyrase和hTE盯在急性白血病细胞和正常人骨髓单个核细胞中的表达呈正相关(二0.454，P<0.001)。tankyrase一2在急性白血病细胞中的表达(0.00493，2.4sx一。一4一0.0443)与正常人骨髓单个核细胞(0 .00277，2.60 x 10-4一0.0!22)中的表达无显著性差异(P刃.056)，在ANLL和ALL细胞中的表达无显著性差异(P二0.902)。tankyrase一2和hTE盯在急性白血病细胞和正常人骨髓单个核细胞中的表达无显著相关性(r=0.179，P=0.1引)。本研究还将30例急性白血病按初诊时外周血白血病总数大于100xl扩和小于!00x!00分为高细胞性白血病和非高细胞性白血病两组。hTERT和4个端粒结合蛋白在两组中的表达均无显著性差异。提示端粒酶活性及4个端粒结合蛋白的表达水平与高细胞性白血病无关。本研究根据白血病细胞染色体核型分析分为染色体为异常核型和正常核型两组，结果hTERT和4个端粒结合蛋白在两组中的表达均无显著性差异。 第二部分:由于4个端粒结合蛋白中唯有TRFI在急性白血病细胞中的表达是下降的，为了探究其表达减少的原因，本研究进一步检测了10个恶性血液病细胞株中TRFI mRNA的表达及突变情况。恶性血液病细胞株包括髓细胞系白血病细胞株K562、HL一60、U937、NB4、T日P一l、HEL、Dami;T淋巴细胞性白血病细胞株6T一CEM、Jurkat和B细胞淋巴瘤细胞株Raji。荧光定量R1一PCR检测TRFI在恶性血液病细胞株细胞中的表达(0.0338，0.0108~0.0749)明显低于正常人骨髓单个核细胞(0.0493，0.0369~0.128)(P=0.004)。其中3个淋系恶性血液病细胞株细胞中TRFI的表达(0.0447士0.0280)低于经单更多还原

【Abstract】 Many studies have reported telomerase is tight-related with malignant hematological diseases.Telomere shortens and telomerase activation was found both in malignant hematological cell line cells and leukemia patients’ samples. However, the mechanism of telomerase activation is unclear. The regulation of telomerase activity could be explained by many aspects, telomere binding protein is one of the new one. Since the first telomere binding protein Telomere Repeat Binding Factor (TRF1) was reported in 1995, about 10 proteins have been reported. They located around telomere, participate in many important cell activities as maintaining telomere structure, mitosis regulation, cell cycle regulation and DNA damage repair etc. Among them, 4 proteins were reported regulate telomerase activity, they are TRF1, Pinxl, tankyrase and tankyrase-2. Here, TRF1 and Pinxl are inhibitors of telomerase activity, while tankyrase and tankyrase-2 are enhancers of telomerase activity. Their expressions and relation with telomerase in acute leukemia cells is unclear.In this study, mRNA expressions of these 4 telomere binding proteins in AL cells were detected and their relations with the mRNA expression of telomerase reverse transcriptase gene (hTERT) were analyzed at first. Then the expression and mutation of TRF1 in malignant hematology cell line cells was analyzed. After that a further study on the telomerase activity and hTERT mRNA expression along with 4 telomere binding proteins’ expression during the differentiation of NB4 cells induced by ATRA was performed to analyze the relation of telomere binding proteins with telomerase during differentiation, so as to indicate the possible mechanism of telomere binding proteins’ effects on telomerase. Expression of 4 telomere binding proteins in leukemia cells have not been reported, nor have the studies on the mutation analysis of TRF1 in malignant hematological cell line cells and the studies of the expression of telomere binding proteins and telomerase activity during differentiation of NB4 cells were reported.This paper was divided into three parts.Part I : Real-time quantitative RT-PCR with fluorescence probe hybridization was used todetect the expression of 4 telomere binding proteins in 30 samples of AL patients, analyze their relation with hTERTmRNA expressions.The 30 AL cases are all enrolled at their first diagnose, 18 were male and 12 female, average age is 32, 17 samples were of acute non-lymphocytic leukemia (ANLL) while 13 of acute lymphoblastic leukemia (ALL), 10 patients’ peripheral WBC was above lOO 10~9 /L while 20 were below 100 109 /L at diagnose, 14 were with abnormal karyotype and 16 with normal karyotype.The expression of hTERTmRNA in AL samples (0.00244, 0-0.0320) is significantly higher than in normal mononuclear cells (2.79X10-4, 0-0.0078)( P<0.001). Its expression has no significant difference between ANLL and ALL cells (P=0.742) . TRF1 mRNA expression in AL samples(0.0171, 0.0127-0.193)is lower than that in normal mononuclear cells(0.0457, 0.00839-0.262) (P =0.019). Its expression in ANLL (0.0126, 0.0127-0.0546) is lower than that in normal mononuclear cells (P<0.001) , but its expression in ALL cells (0.0745, 1.92 l0-6-0.193) has no significant difference with that in normal mononuclear cells(P=0.761 ).TRF1 expression in ANLL is significantly lower than that in ALL cells( P=0.001). The expression of TRFI mRNA in AL samples and normal mononuclear cell samples has no significant correlation with hTERTmRNA expression(r=-0.173, P=0.207) . Pinxl mRNA expression in AL samples (0.00312, 5.42 l0-4~0.024) is significantly higher than that in normal mononuclear cells (7.89 l0-4, 0-0.00863) (P=0.002). Its expression in ANLL and ALL cells has no significantly differences (P=0.869) . The expression of Pinxl mRNA in AL samples and normal mononuclear cell samples has significant positive correlation with hTERTmRNA expression (r=0.296, P=0.028) . tankyrase mRNA expression in AL(0.0209, 0.0106-0.0997) is significantly higher than that in normal mon更多还原