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目的 通过检测骨肉瘤组织中p2lWAF1/CIP1基因的DNA序列变化,探讨p21WAF1/CIP1基因的序列、基因突变与骨肉瘤表型的关系。方法 采用PCR方法扩增骨肉瘤p2lWAF1/CIP1基因外显子。采用sscP方法检测骨肉瘤p21WAF1/CIP1基因DNA缺失、重排和突变。采用双脱氧核苷酸末端终止法进行DNA的直接测序。结果 DNA 序列分析结果显示:骨肉瘤中p21WAF1/CIP1基因第三外显子(exon3)的cDNA全长序列的609位碱基处发生C →T改变,发生率为44.4%(16/36);健康无骨肉瘤家族史者外周血中同样发现p21WAF1/CIP1基因第三外显子的cDNA全长序列的609位碱基处由C→T 改变,发生率为80%(8/10)。结论 p21WAF1/CIP1基因的突变在人类骨肉瘤中是十分少见的。本实验第一次对黄种人骨肉瘤中的p21WAF1/CIP1基因的DNA多态性位点进行了定位,这个位点可能会对今后对该基因的研究提供有意义的参考。
Objective To investigate the relationship between the sequence of p21WAF1 / CIP1 gene and the phenotype of osteosarcoma by detecting the DNA sequence changes of p21WAF1 / CIP1 gene in osteosarcoma. Methods The exon of p2lWAF1 / CIP1 gene was amplified by PCR. The sscP method was used to detect DNA deletion, rearrangement and mutation in p21WAF1 / CIP1 gene of osteosarcoma. Direct DNA sequencing was performed using the dideoxynucleotide termination method. Results The results of DNA sequencing showed that C → T changes occurred at base 609 of the full length cDNA of exon3 of p21WAF1 / CIP1 gene in osteosarcoma with a rate of 44.4% (16/36). In the peripheral blood of healthy non-osteosarcoma family members, the 609 base sequence of the third exon of p21WAF1 / CIP1 gene was also found to be changed from C → T to 80% (8/10). Conclusion The mutation of p21WAF1 / CIP1 gene is very rare in human osteosarcoma. This experiment for the first time in human osteosarcoma P21WAF1 / CIP1 gene DNA polymorphism loci, this site may provide future research on the gene to provide a meaningful reference.