人骨肉瘤p21^(WAF1)基因表达及其DNA序列分析

Expression of p21^(WAF1) gene and DNA sequencing in human osteosarcoma

目的 检测人骨肉瘤组织中 p2 1WAF1基因的表达及其DNA序列变化。方法 采用原位杂交及免疫组化法检测p2 1WAF1mRNA及p2 1蛋白的表达 ,SSCP方法检测骨肉瘤 p2 1WAF1exon3DNA突变 ,双脱氧核苷酸末端终止法进行DNA的直接测序。结果 ①免疫组织化学结果显示 :p2 1蛋白表达阳性率在骨肉瘤中为17 7% (8/ 4 5 ) ;在骨纤维结构不良中为 5 0 % (5 / 10 )。二者的差异有统计学意义 (P <0 0 5 ) ;②原位杂交结果显示 :p2 1WAF1mRNA表达阳性率在骨肉瘤中为 4 2 % (19/ 4 5 ) ;在骨纤维结构不良中为 80 % (8/ 10 )。二者的差异有统计学意义 (P <0 0 5 ) ;③DNA序列分析结果显示 :骨肉瘤中 p2 1WAF1exon3的 6 0 9位碱基处发生C→T突变 ,突变率为 4 4 4 % (16 / 36 )。结论 ①随着骨肿瘤恶性度的升高 ,p2 1WAF1mRNA及p2 1蛋白的表达下降。②p2 1WAF1mRNA在骨肉瘤中的表达对预后有影响。③该实验对中国人群骨肉瘤中的 p2 1WAF1基因的DNA多态性位点进行了新的定位 。

Objective To investigate the expression and DNA sequence status of the p21 WAF1 gene in human osteosarcoma. Methods p21 WAF1 gene in 10 cases of normal and 36 osteosarcoma specimens were examined using polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) silver staining.The PCR product with an abnormal strand was sequenced directly.The p21 WAF1 mRNA and p21 protein of 45 cases of osteosarcoma, 10 normal and 10 fibrous displasia specimens were investigated by in situ hybrization and immunohistochemistry, respectively. Results ①The immunohistochemistry results showed the positive rate of p21 protein was 17.7%(8/45) in osteosarcoma and 50%(5/10) in fibrous displasia of bone, which has significant different(P<0.05); ②The in situ hybrization results showed the p21 WAF1 DNA positive rate was 42%(19/45) in osteosarcoma and 80%(8/10) in fibrous displasia of bone, which has significant different(P<0.05); ③ The sequence analysis showed there was 16 cases undergone mutation on C→T in the 609th point of p21 WAF1 cDNA sequence, and the mutation rate was 44.4%(16/36). Conclusion ①Along with the increase of malignancy, the expression of p21 WAF1 mRNA and p21 protein in osteosarcoma tend to decrease. ② The expression of p21 WAF1 mRNA has a definite value in judging the prognosis in osteosarcoma. ③Our research define the location of p21 WAF1 gene polymorphism of the Chinese osteosarcoma patients and these locations will provide the meaningful reference for the further research of p21 WAF1 gene.