摘要
目的观察ｐ５３基因在肝癌细胞中的正常表达及定位情况。方法根据野生型ｐ５３基因的编码顺序，ＰＣＲ方法扩增突变的ｗｔ－ｐ５３基因，使其３’端的终止密码ＴＧＡ突变为ＴＧＧ；用基因重组技术将其与绿色荧光蛋白（ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｃｅ ｐｒｏｔｅｉｎ， ＧＦＰ）融合，通过真核表达质粒一脂质体介导，导入人高转移肝癌细胞系ＭＨＣＣ９７（ＰＣＲ检测有ｐ５３基因突变）中。用荧光显微镜观察ｐ５３的表达情况。结果ＭＨＣＣ９７细胞中存在ｐ５３基因第２４９位的突变，而低转移入肝癌细胞ＬＤ２０则无此突变。ＤＮＡ顺序测定表明：ｗｔ一ｐ５３基因与ＧＦＰ基因达到编码框架内融合。荧光显微镜显示在空白载体ｐＥＧＦＰ－Ｎ１转染的ＭＨＣＣ９７细胞中荧光布满整个细胞，在转染阳性载体ｐＥＧＦＰ－５３的ＭＨＣＣ９７细胞中荧光仅聚集在细胞核中。结论ｐ５３基因第２４９位上的突变可能与肝癌细胞的转移特性有关；转染的人高转移肝癌细胞系ＭＨＣＣ９７能高效表达ｐ５３蛋白，并定位于细胞核内，与野生型ｐ５３基因的表达方式相同。
Objective To study the normal expression and location of wt-p53 protein on human high-metastasis hepatocellular carcinoma(HCC) cell line. Methods According to the sequence of human wild-type p53, the mp53 cDNA in which stopped codon TGA was mutated to TGG, was amplified by PCR, ligated to EGFP gene, of pEGFP-NI. The re- combinant plasmid was transfected into MHCC97, in which p53 gene was mutated. The expression of human p53 protein was observed by fluorescence microscopy. Results There was a p53 mutation on 249 codon in human high-metastasis hepatocellular carcinoma cell line MHCC97, but no mutation in low-metastasis HCC cells LD20. The two genes of wt-p53 and GFP were fused in codon frame by DNA sequencing. The fluorescence microscopy showed that fluorescence light was spread in all of MHCC97 cells transfected with pEGFP-N1, but fluorescence light was focused on the nucleus of MHCC97 cells transfected with pEGFP-53. Conclusion The p53 mutation on 249 codon may be related to the character of metastasis of HCC cells. The human wild-type P53-GFP fusion protein is expressed in MHCC97 cell line and located in cell nucleus, which is similar to the expression of wt-p53 protein.
出处
《中华肝脏病杂志》
CAS
CSCD
2000年第2期105-107,共3页
Chinese Journal of Hepatology
基金
美国中华医学基金!CMB-95583
