论文部分内容阅读
目的:建立基于分析T细胞受体(TCR)基因重排而确定T细胞-急性淋巴细胞白血病(T-ALL)克隆的新方法,为研究T-ALL中涉及TCR基因位点的染色体易位提供基础。方法:提取1例T-ALL患者外周血单个核细胞(PBMC)的总DNA,利用精细定位的寡核苷酸阵列比较基因组杂交(fine-tiling aCGH)分析样本与对照组基因组DNA的差异,了解不同染色体上可能的断裂点和具体的位点,根据所提供的初步结果,从断裂点涉及的基因序列设计特异引物,采用连接介导PCR(LM-PCR)和序列分析等方法去找出与之发生重排的基因序列。并进一步通过RT-PCR检测TCR基因表达。结果:该例T-ALL患者fine-tiling aCGH结果显示14染色体TCRα/δ基因座出现4个断裂点,分别对应TCR Vδ1、Vδ2、Jδ1和Jδ2基因位点。通过LM-PCR、测序及序列分析,该例T-ALL患者的PBMC中TCR基因涉及Vδ1Dδ2Dδ3 Jδ1、Vδ2Dδ3 Jδ2重排。RT-PCR的结果也验证T-ALL表达该TCR基因重排。结论:结果表明,利用fine-tiling aCGH及LM-PCR技术可以找出TCR基因重排,并且该方法是可靠的,也是发现一些涉及TCR位点的融合基因的一条途径。
OBJECTIVE: To establish a new method for the identification of T-ALL clones based on the analysis of T cell receptor (TCR) gene rearrangements and to provide a basis for the study of chromosomal translocations involving TCR loci in T-ALL basis. Methods: The total DNA of peripheral blood mononuclear cells (PBMCs) of one T-ALL patient was extracted. The difference of the genomic DNA between the sample and control group was analyzed by fine-tiling aCGH Based on the preliminary results provided, specific primers were designed based on the sequence of the gene involved in the breakpoint. Link-mediated PCR (LM-PCR) and sequence analysis were used to find out the possible breakpoints and specific sites on different chromosomes. Gene rearranged gene sequence. TCR gene expression was further detected by RT-PCR. Results: The results of fine-tiling aCGH in T-ALL patients showed that there were 4 breakpoints on TCRα / δ locus of chromosome 14, corresponding to TCR Vδ1, Vδ2, Jδ1 and Jδ2 loci respectively. By LM-PCR, sequencing and sequence analysis, the TCR gene in PBMC of T-ALL patients in this case involved in the Vδ1Dδ2Dδ3 Jδ1, Vδ2Dδ3 Jδ2 rearrangement. The results of RT-PCR also verified T-ALL expression of this TCR gene rearrangement. CONCLUSIONS: The results show that TCR gene rearrangements can be found using fine-tiling aCGH and LM-PCR techniques and that this approach is reliable and is also a means of finding some fusion genes involved in TCR loci.