| 摘要: |
| [目的]探索p53基因突变对细胞正常功能的影响,阐明肝癌的发病机制.[方法]利用PCR产物直接测序的方法,对202例中国肝癌患者p53基因的11个外显子进行突变筛查;利用定点突变的方法构建真核表达载体pCMV-R248W,Western blot检测突变体蛋白R248W在p53缺失型H1299细胞中的表达情况;采用双荧光素酶报告基因检测系统和流式细胞仪,研究R248W突变对转录活性及促凋亡能力的影响.[结果]在其中1例样本的7号外显子处筛查到突变形式为CGG→TGG的点突变,使p53蛋白248位的精氨酸(Arg)突变为色氨酸(Trp),即R248W,突变率为0.495%;在H1299细胞中转染等量的pCMV-p53和pOMV-R248W时.野生型p53与突变体R248W的蛋白表达量相当,但R248W的转录活性及促凋亡能力显著低于野生型p53.[结论]R248W突变可能引起p53蛋白构象的改变,从而影响p53的转录活性及促凋亡能力,使细胞的正常生理功能紊乱,导致肿瘤发生. |
| 关键词: p53 突变 Western blot 转录活性 凋亡 |
| DOI： |
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| 基金项目:国家自然科学基金项目 (30024001) |
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| The cytological function study on a p53 mutant, R248W, in Chinese Hepatocelluar Carcinoma |
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| Abstract: |
| 【Objective】 The study investigated the effect of p53 gene mutation on the normal cellular function and further revealed the pathological mechanism of Hepatocellular carcinoma (HCC).【Method】 11exons of p53 gene were amplified by polymerase chain reaction (PCR) in 202 Chinese HCC patients,and the mutations were detected by direct DNA sequencing;the expression plasmid pCMV-R248W was constructed by site-direct mutagenesis;and the protein expression,the transcriptional activity and the inducing apoptosis ability were respectively investigated by Western blot,Dual Luciferase assay and Flow Cytometry.【Result】 A missense mutation R248W(CGG→TGG),in exon 7 codon 248 of p53 gene was identified in one of the 202 Chinese HCC samples,and the mutation rate was 0.495%.Western blot analysis showed that the p53-null cell line H1299 with equal recombinant pCMV-p53 and pCMV-R248W transfection nearly had the equal protein expression level,however R248W transcriptional activity and the inducing apoptosis ability fell significantly compared with the wild type p53.【Conclusion】 The R248W mutant in Chinese HCC may change the p53's structure,which may affect p53's transcriptional activity and the inducing apoptosis ability.As a result,the normal cytological function is severely impaired,leading to the tumorigenesis. |
| Key words: p53 mutation Western blot transcriptional activity apoptosis |
