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目的:抑癌基因N-myc下游调节基因2(N-myc down stream regulated gene 2,NDRG 2)在抑制肿瘤的发生及进展过程中具有重要作用,本研究旨在区分并鉴定NDRG2基因mRNA的6种可变剪接体。方法:针对NDRG2基因mRNA6种剪接体外显子的差别设计6对特异性引物。分别从正常人脑组织和胶质瘤细胞U251中提取总RNA,反转录为cDNA,利用高保真酶扩增目的剪接体DNA,并根据PCR扩增产物电泳条带的有无和大小初步判断剪接体的型别,最后将PCR产物胶回收进行测序鉴定。结果:利用本实验中设计的引物可以将人脑组织和U251胶质瘤细胞中的NDRG 2基因mRNA剪接体经行分型鉴定,确定了分型引物的可行性。人脑组织和U251细胞中均含有A、B、D和E四种剪接体。结论:利用本研究设计的特异性引物进行PCR扩增,可以判断某种细胞或组织中表达的NDRG2mRNA分子剪接体的型别。有助于未来深入研究抑癌基因NDRG2在肿瘤发生发展中的抑制作用与不同剪接体的表达相关性。
OBJECTIVE: N-myc down stream regulated gene 2 (NDRG2) plays an important role in the inhibition of tumorigenesis and progression. This study aimed to distinguish and identify the expression of NDRG2 mRNA Variable splicing. Methods: Six pairs of specific primers were designed according to the difference of in vitro exon 6 of NDRG2 gene mRNA. Total RNA was extracted from normal human brain tissue and glioma cells U251, respectively, and reverse transcribed into cDNA. The DNA of the target splicing was amplified by high-fidelity enzyme. Based on the presence and absence of size bands, The type of splice body, and finally the PCR product gel recovery sequencing identification. Results: Using the primers designed in this study, the NDRG2 mRNA splicing in human brain tissue and U251 glioma cells was identified by typing and the feasibility of typing primers was confirmed. Human brain tissue and U251 cells contain A, B, D and E four splice bodies. Conclusion: Using the specific primers designed in this study for PCR amplification, we can determine the type of splice body of NDRG2 mRNA expressed in a certain cell or tissue. It is helpful to further study the relationship between the inhibitory effect of tumor suppressor gene NDRG2 in tumorigenesis and the expression of different spliceosomes in the future.