| 摘要: |
| 目的 构建KLF2过表达BGC823和KLF2敲减MGC803胃癌稳转细胞株。方法克隆人源KLF2基因，连接到AgeI/AgeI酶切后GV358载体上，构建GV358-KLF2重组质粒，转染293T细胞，包装慢病毒；构建KLF2shRNA，连接到BamHI和EcoRI双酶切后的PHBLV-U6-ZSGreen-Puro载体上，经鉴定后转染293T细胞。采用RT-PCR及Western印迹法检测稳转细胞株中KLF2的表达。结果利用慢病毒介导，重组质粒GV358-KLF2和KLF2-shRNA转导入BGC823细胞、MGC803细胞并稳定表达。RT-PCR和Western印迹法结果均显示过表达稳转株KLF2表达量明显升高（P＜0.05）,敲减稳转株KLF2表达量明显下降（P＜0.05）。结论成功构建了KLF2过表达BGC823细胞株和KLF2敲减MGC803细胞株，为后续KLF2功能实验奠定了基础。 |
| 关键词: KLF2基因 稳转株 敲减稳转 BGC823细胞 MGC803细胞 |
| DOI：10.16118/j.1008-0392.2017.05.004 |
| 投稿时间：2017-04-12 |
| 基金项目:上海市自然科学基金（16ZR1429100） |
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| Establishment of gastric cancer cell lines with over-expressed KLF2 and knockdown KLF2 |
| WANG Chun-mei,CHEN Jin-lian |
| (Dept. of Gastroenterology, East Hospital, Tongji University, Shanghai 200120, China;Dept. of Gastroenterology, South Campus of Sixth Peoples Hospital, Shanghai Jiaotong University, Shanghai 201499， China) |
| Abstract: |
| Objective To establish gastric cancer cell line with over-expressed KLF2 and knockdown KLF2. MethodsThe recombinant plasmid GV358-KLF2 was constructed by cloning human KLF2 gene into AgeI/AgeI enzyme-digested GV358 vector, then the GV358-KLF2 plasmid was transfected into 293T cells to pack lentivirus. Three pairs of synthesized shRNAs were inserted into BamHI and EcoRI enzyme-digested PHBLV-U6-ZSGreen-Puro vector, and packed into lentivirus. The expression of KLF2 was detected by RT-PCR and Western blot. ResultsRecombinant plasmids GV358-KLF2 and KLF2-shRNA were successfully transfected into BGC823 and MGC803 respectively, and expressed stably. The results of RT-PCR and Western blotting showed that the expression of KLF2 was increased in KLF2-overexpressed BGC823 cells (P＜0.05), and decreased in KLF2-knockdown MGC803 cells (P<0.05). ConclusionKLF2-overexpression BGC823 cell line and KLF2-knockdown MGC803 cell line have been successfully established, which may be used for the study of KLF2 function. |
| Key words: Krüppel-like factor 2 overexpression cell line knockdown cell line BGC823 cell MGC803 cell |
