14株肿瘤细胞P53基因突变的检测

Detection of P53 gene mutations in 14 tumor cell lines

目的：检测14株肿瘤细胞P53基因的突变情况。方法：根据P53基因5～8外显子之间的内含子序列设计引物,分别对人肺癌、脑胶质瘤、肝癌、胃癌、前列腺癌、乳腺癌、结肠癌、脉络膜恶性黑素瘤、视网膜成细胞瘤等9种肿瘤14株细胞的P53基因5、6、7、8的外显子进行PCR扩增反应。琼脂糖凝胶电泳鉴定PCR产物;DNA测序,并与正常的DNA序列比较;抽提肿瘤细胞蛋白进行Western blotting检测P53蛋白的表达。结果：14株肿瘤细胞P53基因热变区5、6、7、8外显子的扩增产物经电泳鉴定与预期相同。DNA测序结果表明,14株肿瘤细胞中8株存在P53基因5～8外显子的突变,其中肺癌细胞H1299、肝癌细胞Hep3B、肝癌细胞SMMC7721、脉络膜恶性黑素瘤细胞OCM-1检测到以前未报道过的突变;突变主要发生在外显子的编码序列,多数为单个碱基替换导致的错义突变,也有部分表现为同义突变;2株正常细胞未检测到突变。Western blotting检测显示,有基因突变的8株肿瘤细胞中仅6株有P53蛋白表达;所有P53基因未突变的肿瘤细胞株和正常细胞株均未检测到P53蛋白。结论：检测的14株肿瘤细胞中有8株细胞P53基因热变区存在突变,其中4株检测到以前未报道过的突变;2株正常细胞未发现P53基因突变。

Objective：To investigate P53 gene mutations in 14 tumor cell lines. Methods： Primers were designed according to the intron sequences locating between exons 5-8 of P53 gene for amplification of P53 gene exon 5-8 of the 14 tumor cell lines from 9 kinds of human carcinoma, namely, the lung cancer, gloma, hepatocarcinoma, gastric carcinoma, prostate carcinoma, mammary carcinoma, colonic carcinoma, choroidal melanoma, and retinoblastoma. The PCR products were subjected to electrophoresis; DNA sequencing was performed for the amplified product and the results were compared with wild-type P53 gene. Meanwhile, the P53 protein of 14 tumor cell lines was extracted for Western blotting analysis. Results： The products of exons 5-8 amplified by PCR were identical to that expected. The results of DNA sequencing showed that 8 of 14 tumor cell lines had P53 gene mutation in exons 5-8. Novel mutations were found in 4 tumor cell lines, including human lung cancer cell H1299, hepatocacinoma cell Hep3B and 7721, and choroidal melanoma cell OCM-1. The mutations mainly existed in the coding areas of the exons; most of them were missense mutations due to single base replacement; some others were silent mutations; and no mutations were found in the 2 normal cell lines. The results of Western blotting showed that only 6 of 8 mutant lines had P53 protein expression, which was not found in the non-mutant lines and the normal cells. Conclusion： We have identified 8 lines with mutant P53 genes among the 14 tumor cell lines, novel mutations are found in 4 tumor cell lines and none in the two normal cell lines.