| 摘要: |
| 目的:利用稳定表达人miR-122前体的表达载体,转染低表达miR-122的人肝癌细胞系HepG2细胞,获得过量表达miR-122的稳定转染细胞系,探讨miR-122对肝脏细胞脂代谢的影响及其机制。方法:对已构建完成的表达质粒pEZX-hsa-mir-122和对照质粒pEZX-mir-control进行酶切鉴定,对酶切正确的克隆进行DNA测序,测序正确的克隆应用脂质体试剂转染,绿色荧光蛋白表达监测转染效率,应用嘌呤霉素筛选稳定转染细胞系,实时定量PCR在RNA水平鉴定miR-122的表达,蛋白质印迹法从靶分子水平验证miR-122的抑制功能,尼罗红染色观察细胞内脂质。结果:pEZX-hsa-mir-122质粒成功转染HepG2细胞,质粒携带的miR-122前体序列整合到细胞基因组中,获得过量表达miR-122的HepG2细胞株,细胞内成熟体miR-122可抑制靶基因的翻译,细胞内脂质明显增加。结论:人miR-122前体序列可整合在HepG2细胞基因组中,过量表达miR-122的HepG2细胞出现脂质代谢异常。 |
| 关键词: 人miR-122 稳定转染 脂代谢 嘌呤霉素 HepG2细胞 |
| DOI：10.3724/SP.J.1008.2009.0825 |
| 投稿时间：2009-02-16修订日期：2009-06-17 |
| 基金项目:国家自然科学基金（30671829）. |
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| Establishment of hsa-mir-122 stably transfected-HepG2 cell line and its lipid metabolism characteristics |
| ZHANG Ya-nan△,WANG Fang△,YANG Fu,YIN Yi-xuan,ZHANG Ling,SUN Shu-han* |
| (Department of Medical Genetics,College of Basic Medical Sciences,Second Military Medical University,Shanghai 200433,China) |
| Abstract: |
| Objective:To establish a HepG2 cell line stably overexpressing hsa-mir-122 by transfecting the HepG2 cells with pEZX-hsa-mir-122 vectors,so as to observe the influence of miR-122 on the lipid metabolism in the liver cells.Methods:The constructed pEZX-hsa-mir-122 and pEZX-mir-control plasmids were identified by restriction enzyme digestion and the right clones by sequencing were subjected to sequencing.And the right clones by sequencing were transfected by LipofectamineTM.The transfection efficiency was assessed by green fluorescent protein expression.The stably transfected cell lines were screened by Puromycin.The expression of miR-122 was examined by real-time PCR.The inhibitory function of miR-122 was testified by Western blotting assay.Nile red was used to observe the content of lipid in HepG2 cells stably overexpressing hsa-mir-122.Results:HepG2 cells were successfully transfected with pEZX-hsa-mir-122 and pEZX-mir-control plasmids.The precursor miRNA sequences were integrated into the genome of HepG2 cells stably overexpressing miR-122.Compared with the control HepG2 cells,the HepG2 cells overexpressing miR-122 contained more lipid in the cytoplasm.Conclusion:The miR-122 precursor sequence can intergrate into the genome of HepG2 cells.The increase of miR-122 level in HepG2 cells can result in abnormal metabolism of lipid. |
| Key words: hsa-mir-122 stable transfection lipid metabolism puromycin HepG2 cell |
