医学分子生物学杂志2011，Vol.8Issue(5)：386-391,6.DOI:10.3870/j.issn.1672-8009.2011.05.003
MEKK3稳定表达细胞株的建立及其对A549细胞增殖的影响
Establishment of a Cell Line Stably Expressing MEKK3 and Effect of MEKK3 on Proliferation of A549 Cells
摘要
Abstract
Objective To construct eukaryotic expression vector pcDNA3. 1/hygro ( + ) -MEKK3, establish cell clones stably expressing MEKK3 and study the effect of MEKK3 on proliferation of lung adenocarcinoma cells. Methods Total RNA was extracted from A549 lung cancer cells. MEKK3 cDNA was amplified by RT-PCR, and inserted into pcDNA3. 1/hygro ( + ) vector to construct pcDNA3. 1/hygro ( + ) -MEKK3. The recombinant plasmid was transfected into A549 cells by lipofectamine. The stable transfectants were obtained with hygromycin B. Western blot and real-time PCR were used to detect the expression of MEKK3 protein and mRNA. MTT assay was used to evaluate the effects of MEKK3 on proliferation of A549 cells. Results The recombinant expression vector pcDNA3. 1/ hygro ( + ) -MEKK3 was successfully constructed. The expression of MEKK3 protein was significantly higher in A549 cells transfected with the MEKK3 expression vector than in those transfected with empty vector or untransfected A549 cells. In addition, the expression of MEKK3 mRNA was significantly higher in stable transfectants with the MEKK3 vector than in those with empty vector or in untransfected cells ( P < 0. 05) , and there was no significant difference between the latter two groups (P > 0. 05) .By MTT assay, we found that the proliferation activity of A549 cells in stably transfected group with the MEKK3 expression vector (A570 =0. 8761 ± 0. 0745) was significantly stronger than in those with empty vector (A570 =0. 5828 ±0. 0703) or inuntransfected cells (A570 =0. 5849 ±0. 0352) (P <0. 01) , and there was no significant difference between the latter two groups (P > 0. 05) . Conclusion MEKK3 can increase proliferation of lung adenocarcinoma cells.关键词
MEKK3基因/基因克隆/基因转染/荧光实时定量PCR/四唑盐比色测定Key words
MEKK3 / gene cloning/ gene transfection/ Real-time PCR/ MTT assay分类
生物科学引用本文复制引用
沈瑞明,兰风华,钱凤英,王志红,董荔红,黄俏佳..MEKK3稳定表达细胞株的建立及其对A549细胞增殖的影响[J].医学分子生物学杂志,2011,8(5):386-391,6.基金项目
福建省自然科学基金(No.2009J01181),南京军区医药卫生科研基金(No.08MA100) （No.2009J01181）
