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目的:检测原代肝癌组织以及肝癌细胞系中p53的突变位点和频率,了解p53突变与肝癌发病和治疗的关系。方法:设计了3对引物,采用PCR技术扩增p53的5—8外显子,然后进行DNA测序鉴定;共测定了两个肝癌细胞系(BEL-7402和2.2.15)以及两例原代肝癌组织。其中5和6外显子用一对引物串联扩增出来,上游引物序列为:5′-GGAATTCCTCTTCCTGCAG-3′,下游为:5′-GGAATTCAGTT GCAAACCAGAC-3′。然后从5′和3′端分别测定5和6外显子序列。其它外显子则分别扩增测序。结果:在肝癌细胞系和原代肝癌组织均未发现p53基因5—8外显子突变。但扩增BEL-7402细胞DNA的5—6外显子时,除得到5—6外显子串联产物外,还得到了一段新的DNA片段,长度为200bp左右。以5—6外显子串联产物为模板做PCR则不能扩增出上述新片段,说明新片段并非来源于5—6外显子。为此对纯化的新片段重复测序两次,结果完全一致,确定的一段DNA序列(100bp左右)列出如下:~(20CA)GCA CAT GAT GTG AAA AAC TGC CTG CTT CCG AGA AAA TGG GGG TTT CCA AAA CAA TGG AAG TGA TTC TTT GCA NAA GGC TAC TAT TGG GTG GTA CTT TCT TGT~(123)。结论:在检测的4例肝细胞癌中未发现p53的5—8外显子突变,此项工作对我室将要进行的肝癌基因治疗的病例筛选具有重要意义。此外在扩增5—6外显子时意外得到一段新DNA序列,分析表明该序列不存在于p53基因之中,值得做进一步鉴定。
Objective: To detect the mutation site and frequency of p53 in primary hepatocellular carcinoma and liver cancer cell lines, and to understand the relationship between p53 mutation and the onset and treatment of liver cancer. METHODS: Three pairs of primers were designed, and 5-8 exons of p53 were amplified by PCR and then sequenced by DNA sequencing. Two hepatocellular carcinoma cell lines (BEL-7402 and 2.2.15) and two primary samples were determined. Liver cancer tissue. Exon 5 and exon 6 were amplified in tandem using a pair of primers. The sequence of the upstream primer was: 5′-GGAATTCCTCTTCCTGCAG-3′ and downstream was: 5′-GGAATTCAGTTGCAAACCAGAC-3′. The 5 and 6 exon sequences were then determined from the 5’ and 3’ ends, respectively. Other exons were amplified and sequenced separately. RESULTS: No mutations in exon 5-8 of p53 gene were detected in the hepatoma cell lines and primary hepatoma tissues. However, when the exons 5-6 of DNA of BEL-7402 cells were amplified, in addition to the 5-6 exon tandem product, a new segment of DNA was obtained with a length of about 200 bp. Using the 5-6 exon tandem product as a template to perform PCR could not amplify the above new fragment, indicating that the new fragment is not derived from the exon 5-6. For this purpose, the purified new fragment was repeatedly sequenced twice and the results were completely identical. The determined DNA sequence (about 100 bp) is listed as follows: ~(20CA)GCA CAT GAT GTG AAA AAC TGC CTG CTT CCG AGA AAA TGG GGG TTT CCA AAA CAA TGG AAG TGA TTC TTT GCA NAA GGC TAC TAT TGG GTG GTA CTT TCT TGT~(123). Conclusion: No mutations in exon 5-8 of p53 were found in the 4 cases of hepatocellular carcinoma detected. This work is of great significance to the screening of cases of liver cancer gene therapy to be performed in our department. In addition, a new DNA sequence was unexpectedly amplified during amplification of 5-6 exons. Analysis showed that the sequence was not present in the p53 gene and deserved further identification.