摘要
目的观察血红素加氧酶-1（remeoxygehase-1，HO-1）活性改变对人肺腺癌A549细胞失巢凋亡的影响。方法将野生型HO-1（wtHO-1）和失活突变体mHO-1G143H基因与质粒pcDNA3-1（＋）连接，构建野生型和突变型HO-1真核表达载体pcDNA3．1（＋）-wtHO-1和pcDNA3．1（＋）-mHO—1G143H。利用脂质体介导的方法将构建好的重组载体转染人肺腺癌A549细胞构建稳转细胞系，以空载体转染作为对照组。通过G418筛选建立稳定表达野生型和突变型HO-1的人肺腺癌A549细胞系。利用RT—PCR及Western印迹方法检测各稳转细胞系中HO-1mRNA和蛋白的表达水平。悬浮培养上述稳转细胞系，建立失巢凋亡模型：应用流式细胞术检测细胞凋亡情况。结果与空载体对照组相比，野生型HO-1过表达细胞系中，细胞失巢凋亡率明显下降；突变型HO-1过表达细胞系中，细胞失巢凋亡率增高。结论HO-1过表达可能抑制肺腺癌A549细胞的失巢凋亡能力，这种作用可能与HO-1催化活性相关。
Objective To determine the effect of heme oxygenase-1 on anoikis in human lung adenocarcinoma cell lines A549. Methods To construct the eukaryotic expression vectors of wild- type HO-1 and HO-1 G143H mutant, named by pcDNA3.1 （ ＋ ）-wtHO-1 and pcDNA3.1 （ ＋ ）- mHO-1G143H. A549 cells were transfected with wtHO-1 and mHO-1 using lipofectamine 2000, empty vector transfected as a control group. Stable transfected cells were selected by G418. The ex- pression of HO-1 mRNA and protein were detected by RT-PCR and Western blot. Suspension cul- tures of the stable transfected cell lines to establish the model of anoikis and detect the apoptotic a- bility of A549 by Flow Cytometer. Results Compared to the empty group, overexpression of wtHO-1 decrease the apoptosis of A549 cells after detached culture for 72h. On the contrary, over- expression of mHO-1 increase the rate of anoikis of A549 cells. Conclusion Overexpression HO-1 inhibit anoikis of lung adenocarcinoma A549 cells which may be the result of catalytic activity of HO-1.
出处
《医学分子生物学杂志》
CAS
CSCD
2012年第5期338-343,共6页
Journal of Medical Molecular Biology
基金
国家自然科学基金（No.81171997,No.81000933）,中国博士后科学基金（No.2012M520035）,黑龙江省卫生厅基金（No.2009-211）
