【作者】 陈沛帅；

【导师】 陈子兴；

【作者基本信息】 苏州大学 ， 内科学， 2008， 硕士

【摘要】 目的及意义DNA双链断裂(double-strand break, DSB)是DNA在受到各种损伤因素作用后最严重的损伤方式之一,可导致基因变异或细胞死亡。DSB发生时,机体可通过同源重组(homologous recombination, HR)和非同源末端连接(non-homologous DNA end joining, NHEJ)两条途径对其进行修复,而后者是高级哺乳动物修复DSB的主要途径,Ku70是其核心因子之一。本研究拟从基因转录水平和蛋白水平检测慢性粒细胞性白血病(Chronic myeloid leukaemia,CML)Ku70在初诊CML患者细胞中的表达,并对Ku70和BCR/ABL融合基因表达水平进行相关性分析,以探讨Ku70表达及NHEJ在CML发病中的作用,为阐明CML的确切发病机制提供新的线索。方法1. Ficoll分离初诊CML各期患者及正常供体骨髓单个核细胞,取2×107个单个核细胞用于后续实验。2. RT-PCR和实时定量RT-PCR(RQ-RT-PCR)检测初诊CML细胞和正常供体骨髓细胞中Ku70基因在转录水平的表达。3. RQ-RT-PCR检测初诊CML细胞中BCR/ABL融合基因在转录水平的表达。4. Western Blot印迹法检测初诊CML和正常供体骨髓细胞Ku70蛋白的表达。以Ku70与内参TBP(TATA Box Binding Protein)的灰度比对Ku70蛋白的表达水平进行半定量分析。5.采用SPSS13.0分别分析以下因素之间的相关性:(1)初诊CML中BCR/ABL与Ku70在转录水平的表达量;(2)初诊CML中BCR/ABL mRNA与Ku70蛋白的表达量;(3) Ku70在转录与蛋白水平的表达量。结果1. RT-PCR检测Ku70基因转录的表达:24例正常供体骨髓细胞中几乎看不到Ku70基因mRNA的表达;CML各期细胞中的Ku70基因表达均明显高于正常对照组,同时12例CML急变加速期细胞中Ku70基因的表达也明显高于CML慢性期。2. RQ-RT-PCR检测Ku70基因转录的表达:CML各期细胞中Ku70基因的表达量(544.63±185.71 Ku70拷贝/10000β-Actin拷贝)显著高于正常对照组(31.08±8.41,P=0.039),同时CML急变加速期细胞中Ku70基因的表达量(1103.31±645.62)远高于正常对照组和CML慢性期细胞(97.69±40.73,P<0.01),而慢性期细胞中Ku70基因的表达与正常细胞之间的差异无统计学意义(P>0.05)。3. Western Blot印迹法检测Ku70蛋白的表达:24例正常供体骨髓细胞中Ku70蛋白与TBP灰度比为(0.10±0.09),27例CML细胞中为(0.40±0.21),明显高于前者(P<0.001)。其中,12例急变加速期CML细胞为(0.57±0.22),15例慢性期CML细胞为(0.27±0.04),二者之间差异有统计学意义(P<0.001)。4.相关性分析结果:(1)初诊CML各期细胞中BCR/ABL融合基因与Ku70基因在mRNA水平的表达呈正相关关系(r=0.573,P=0.002);(2)初诊CML各期细胞中BCR/ABL融合基因表达与Ku70蛋白表达呈正相关关系(r=0.705, P<0.001);(3) Ku70基因在mRNA和蛋白水平的表达亦呈显著的正相关性(r=0. 808, P<0.001)。结论1. CML各期细胞中Ku70基因的表达高于正常对照组,提示以Ku70为核心因子的NHEJ修复途径在CML中被过度激活。2. CML急变加速期细胞中Ku70基因在mRNA和蛋白水平的表达量均远高于正常对照组和CML慢性期细胞,而慢性期细胞中Ku70在mRNA和蛋白水平的表达与正常细胞间无显著差异。提示NHEJ途径的过度激活在CML自慢性期向急变加速期的演变过程中发挥了重要的作用。3. CML细胞中Ku70基因和蛋白的表达与BCR/ABL融合基因的水平呈正相关性。说明CML细胞中BCR/ABL融合基因的高表达与NHEJ途径过度激活呈相同趋势,提示在CML细胞中BCR/ABL的表达与NHEJ途径修复DSB密切相关。4.在CML细胞中Ku70基因与Ku70蛋白的表达量呈正相关性,说明在CML细胞中Ku70在mRNA和蛋白水平表达量的变化是同步的,提示CML细胞通过NHEJ修复DSB可能同时发生在基因水平和蛋白水平。更多还原

【Abstract】 ObjectiveThe DNA double strand breaks (DSB) in mammalian cells, caused either by ionizing radiation, radiomimetic drugs, or occurring during gene rearrangements, are predominantly repaired by a process called non-homologous DNA end joining (NHEJ). The NHEJ pathway contains several key proteins to undertake its function, among which Ku70 played a pivotal role. As a common malignant disease of hematological system, chronic myeloid leukemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. As reported, there were some correlations between the expression of CML special fusion gene BCR/ABL and the high efficiency of the DSB repairs. The present study investigated the expression of the gene Ku70 and the protein Ku70 in leukemia cells of CML patients at different clinical stage. To reveal the correlation among the expression of the gene Ku70, the protein Ku70 and BCR/ABL in cells of CML patients.Methods1. Bone marrow cells were collected from GM-CSF mobilized normal adults and preliminary diagnosed chronic myeloid leukemia (CML) patients, Separated their mononuclear cell by Ficoll, took 2×107 for the experiment. Detect the living cell rate of bone marrow prepare using trypan blue, make sure WBC above 95% by Wright staining.2. Extract RNA of each group by trizol one step, purify and reverse transcripted to cDNA. Detected the expression of gene Ku70 in CML cells and mononuclear cells of normal BM by RT-PCR and RQ-RT-PCR.3. Extract nuclear protein of each group, quantified by Bradford, Investigated the expression of protein Ku70 in nuclear protein extracted from CML cells and mononuclear cells of normal BM by Western Blot, using TBP(TATA Box Binding Protein) as the internal control.Get the result as the ratio of gray scale of Ku70 over TBP. 4. Took tales doses cDNA from each group, detected the expression of the fusion gene BCR/ABL by TaqMan probe Real Time PCR, using ABL as the internal control.5. Investigated the correlation in cells of preliminary diagnosed CML patients using software SPSS version 13.0: (1) between the expression of the gene Ku70 and BCR/ABL;(2) between the expression of the protein Ku70 and BCR/ABL; (3) between the expression of the gene Ku70 and the protein Ku70.Results1. Qualitative analysis by RT-PCR and quantitative analysis by RQ-RT-PCR of gene Ku70 showed: the expression of Ku70 in total CML cells (544.63±185.71Ku70 copies/10000β-Actin copies) was manifestly higher than in normal BM cells (31.08±8.41Ku70 copies/10000β-Actin copies,P=0.039). Meanwhile, its expression in CML cell of acute phase (1103.31±645.62Ku70copies/10000β-Actin copies) was obviously higher than that of chronic phase (97.69±40.73Ku70 copies/10000β-Actin copies, P<0.01). However, there was no significant difference between the expression of gene Ku70 in CML chronic phase and in normal BM cells. The sequencing of Part of the PCR product was completely matched with gene Ku70 in gene bank of NCBI.2. The gray scale of protein Ku70 over TBP of 24 normal BM cells were (0.10±0.09), while which of 27 CML cases were (0.40±0.21,P<0.001). Among these 27 CML cases, 12 were in acute phase, 15 were in chronic phase, the gray scales of Ku70 were significantly different from each other (mean 0.57±0.22 vs. 0.27±0.04, P<0.001).3. The analysis of dependability showed: there were positive correlations of BCR/ABL when compared with the expression of the gene Ku70 (r=0.573 P=0.002) while with it of the protein Ku70 (r=0.705 P<0.001) in cells of CML, meanwhile, the correlations between the expression of the gene Ku70 and the protein Ku70 (r=0. 808, P<0.001).Conclusions1. The gene and protein of Ku70 were expressed significantly higher in CML than in normal BM cells. Meanwhile, its expression level in CML (acute phase) was markedly higher than that in CML (chronic phase). Suggested that Ku70 was higher expressed in CML cells, means DNA in CML cells was mainly damaged by DSB, and this kind of DNA damage was repaired by NHEJ pathway predominantly. This conclusion made us to presume that pathogenesy of CML may be DNA damage and its unfaithful repair. A new strategy of the study on nosogenesis and its gene therapy of CML was in front of us.2. The analysis of dependability showed there were positive correlations among the expression of fusion gene BCR/ABL, the gene Ku70 and the protein Ku70 in cells of CML. Suggest that through the NHEJ pathway repairing DSB played a very important role in the genesis and progression of CML. Meanwhile, it suggested that the DNA damage and its unfaithful repair in CML cells have happened in both gene and protein level. So it provided more accurated localization of the study on the DNA repair in CML cells.3. But there is still no direct and exact evidence of the mechanism about how DSB induced the generation of CML and its fusion gene BCR/ABL, it’s meaningful and necessary to study this subject furtherly.更多还原