【作者】 张轶文；

【导师】 王少元； 林旭；

【作者基本信息】 福建医科大学 ， 内科学， 2005， 硕士

【摘要】 白血病是血液系统最常见的恶性肿瘤。白血病的发生是一个涉及多基因、多阶段的复杂过程,因此,全面分离与分析参与白血病发生的生物活性物质,揭示这些生物活性物质的功能与途径,对于系统研究与阐明白血病发生的分子机制尤为重要。遗传家系为我们提供了很好的样本来源,是获得疾病相关致病基因的一条重要途径。【目的】分离家族性急性髓系白血病患者骨髓中差异表达的基因,在基因水平上探讨急性白血病发生发展的分子机制。【方法】采用Super SMART和SSH 技术,以患者骨髓作为Tester,正常人骨髓作为Driver,分离患者骨髓细胞中差异表达的基因片段;连接T 载体,转化宿主菌,构建家族性急性髓系白血病cDNA 抑制性消减文库;采用DIG 标记探针的改良Differential Screening技术进行筛选和鉴定;阳性克隆送交测序,BLAST 同源性比对分析,新基因片段提交GenBank。【结果】成功构建了家族性急性髓系白血病cDNA 抑制性消减文库,巢式PCR 进一步使差异表达基因得到了指数扩增。产物连接载体并成功转化TOPO10F’大肠杆菌感受态细胞,使文库中的差异表达基因得到分离。经Differential Screening 技术筛选鉴定,确认28 个基因为阳性差异表达基因。测序结果提交GenBank 进行同源性比对分析,其中17 条与已知基因同源,11 条为未知基因片段。【结论】1. SSH 技术是筛选差异表达基因的有效方法。2. 采用DIG 标记探针替代同位素P³²-dCTP 标记探针,除继承了其高度灵敏度和良好的特异性之外,还避免了放射性污染,简化了实验操作。3. 获得多个与细胞增殖和分化相关的基因,说明急性白血病的发生是一个涉及多基因、多阶段发生的复杂过程。4. 获得11 条在文库中差异表达的新基因EST 片段,并被GenBank 收录,登录号分别为CX129926-7,CV884199,CV884202-3,CV973096-101。更多还原

【Abstract】 Acute leukemia is a common malignant tumour of hematological system. It hasbeen proved by former researches that its pathogenesis relates to multi-genes andmulti-stages. However, it is still not enough evidence to explain it by current researchdata. So, isolating biological active substances in acute leukemia processes andrevealing their functional approaches are very important to understand molecularmechanisms of acute leukemia. The heredity families provide us the better samplesthat are helpful to understand the related virulence genes. 【Objective】To isolategenes expressed differentially from bone marrow cells of familial acute myelgenousleukemia and to explain the molecular mechanisms of the disease at the gene level.【Methods 】Super SMART cDNA synthesis and suppression subtractivehybridization (SSH) were performed to isolate differentially expressed cDNAfragments from patient’s bone marrow cells. cDNA from the patient were used as“tester”, the other from the normal human as “driver”. Subtractive products weredirectly inserted into T/A cloning vector, and then transformed into host bacteria, toset up a subtractive cDNA library of specially or highly expressed genes in familialacute myelogenous leukemia. Those clones were screened and identified by reformeddifferential screening technique which the probes were labeled with DIG. Positiveclones were sequenced and compared with known sequences in the public databasesof GenBank/EMBL/DDBJ using NCBI BLAST for homology analysis. The unknownfragments were then submitted to GenBank. 【Results】Total RNA were extractedand purified in good quality. Double strand cDNA were reverse transcriptedintegratedly, and then cut by RsaⅠinto even length short segments. Ligation wasidentified highly effective. After two hybridizations, a subtractive library ofdifferentially expressed genes in human familial acute myelgenous leukemia wasconstructed successfully by SSH. Nested-PCR was specificial, which amplified thedifferentially expressed genes exponentially. Subtractive products were inserted intothe T/A clone vectors and then transformed into TOPO10F’competent cellssuccessfully, which dispersed differentially expressed genes of the library into a singlecopy. After differential screening and RT-PCR, 28 clones were confirmed to bepositive differentially expressed genes fragments. Sequences were submitted toGenBank for homology analysis to determine what kind of protein they were encoding,and confirm which fragments were new ESTs. Then 17 sequences were a part ofknown genes, and the rests were verified unknown fragments.【Conclusion】(1) SSHis an effective approach to isolate differentially expressed genes. (2) Labeling probeswith DIG was applied in differential screening technology which substituted for theconventional P32-dCTP isotope probes. This technique was not only maintaining thehigh sensitivity and specificity but also averting isotope pollution and simplifyingexperimental procedures. (3) Some genes related to cell proliferation anddifferentiation were acquired by SSH, which proved sufficiently that the occurrence ofacute leukemia was involved in multi-genes and multi-stages. It is a complex process.(4) Eleven novel ESTs related to library were successfully isolated and accepted byGenBank, the access numbers are CX129926-7, CV884199, CV884202-3,CV973096-101, respectively.更多还原