摘要
目的构建pcDNA3．1－RASSFl真核表达载体，并检测其对肝癌细胞系HepG2凋亡的影响。方法应用聚合酶链反应技术从人RASSFlcDNA中扩增出RASSF1基因后，以内切酶XhoI和EcoRI进行双酶切，将其克隆人用相同酶处理的载体pcDNA3．1；将重组质粒pcDNA3．1－RASSF1转染肝癌HepG2细胞，应用Westernblot法检测RASSF1的表达水平；用AnnexinV／PI法检测细胞的凋亡情况。结果酶切和测序结果表明，重组质粒pcDNA3．1．RASSF1构建成功；Westernblot法结果表明转染pcDNA3．1-RASSF1后HepG2细胞中RASSFl表达升高；AnnexinV／PI法用流式细胞术检测凋亡，空白组、pcDNA3．1组和pcDNA3．1-RASSFl组HepG2细胞的凋亡率分别为（5．8±0．42）％、（7．48±0．68）％和（35．1±3．15）％。结论真核表达载体pcDNA3．1-RASSF1构建成功；RASSF1蛋白在HepG2细胞中高表达，并促进细胞凋亡。
Objective To construct pcDNA3.1 RASSF1 eukaryotic vector and observe the influence of RASSF1 on the apoptosis of hepatocarcinoma cell line HepG2. Methods RASSF1 gene was ampli- fied from human RASSF1 cDNA by polymerase chain reaction （PCR） and cloned into pcDNA3.1. The re- combinant plasmid pcDNA3.1 RASSF1 was transfected into hepatocarcinoma HepG2 cell line. The expression of RASSF1 was examined by Western blot. The influence of RASSF1 on the cell apoptosis was meas- ured by Annexin V/PI assay. Results DNA enzyme digestion and sequencing results showed that recombi- nant plasmid pcDNA3.1-RASSF1 was successfully constructed. RASSF1 protein was overexpressed in HepG2 cell line transfected with pcDNA3.1-RASSF1 plasmid. The apoptosis rate of blank, pcDNA3.1 and pcDNA3.1-RASSF1 group was （5.8 ± 0. 42） %, （7.48 ± 0.68 ） % and （ 35.1 ± 3.15 ） %, respectively. Conclusion The pcDNA3.1- RASSF1 eukaryotic vector was successfully constructed, RASSF1 protein overexpression could induce apoptosis in HepG2 cell line.
出处
《中国医师杂志》
CAS
2011年第1期16-18,22,共4页
Journal of Chinese Physician
