pcDNA3.0-CBP真核表达质粒的构建及对肺腺癌SPC-A1细胞的影响

目的 观察外源性CBP基因稳定转染对人肺腺癌SPC-A1细胞体外生长的影响.方法 构建CBP真核表达质粒pcDNA 3.0-CBP,应用pcDNA 3.0-CBP和空载体质粒pcDNA3.0(-),脂质体转染法转染体外培养的人肺腺癌细胞株SPC-A1,G418(800mg/L)筛选出抗性克隆.Western blot检测转染前后CBP蛋白水平变化,噻唑蓝(MTT)比色法分析细胞生长抑制作用,Transwell体外侵袭实验和Wound-healing实验对细胞进行侵袭和迁移能力研究.结果 转染CBP基因的细胞株有目的 基因整合和相应蛋白高表达.MTT检测pcDNA 3.0-CBP转染组活细胞吸光度(0.2787±0.0786)低于未转染组(0.5089±0.1301)和pcDNA 3.0(-)空载体转染组(0.4804±0.1547).pcDNA 3.0-CBP转染组与两对照组吸光度的差异有统计学意义(P<0.01).细胞侵袭实验表明pcDNA 3.0-CBP转染组穿透滤膜数(3.52±0.77)明显少于未转染组(8.17±0.86)和空载体转染组(8.22±1.30),转染组与两对照组侵袭力的差异有统计学意义(P<0.01).细胞迁移实验转染组细胞迁移数(71.30±7.68)明显少于未转染组(119.40±8.38)和空载体转染组(111.41±12.56),转染组与两对照组迁移力的差异有统计学意义(P<0.01).结论 外源性CBP基因稳定转染可抑制人肺腺癌SPC-A1细胞的恶性表型,可能通过对Src家族激酶(SFKs)的负反馈调节抑制肿瘤细胞的增殖、侵袭.

Construction of pcDNA3.0-CBP eukaryotic expression plasmid and its effects on lung adencarcinoma cell line SPC-A1

Objective To investigate the effects of exogenous wild CSK-binding protein (CBP) gene stably transfection on growth of lung adencarcinoma cells in vitro. Methods A recombinant eukary-otic expression plasmid pcDNA3.0-CBP was constructed. Human lung cancer cell line SPC-A1 was trans-fected with pcDNA3.0-CBP or mock transfected plasmid pcDNA3.0 (-) with lipofectamine2000, and cells that expressing CBP stably were screened out by G418 (800 mg/L). Change of CBP protein level was measured by Western-blot, and cell viability was tested by MTT assay. Transwell and wound-healing meth-ads were used to detect the difference of invasion and migration between transfected and non-transfected cells. Results CBP protein level in pcDNA3.0-CBP transfected SPC-A1 cells was significantly higher than that in control SPC-A1 cells and pcDNA3.0 (-) transfected SPC-A1 cells. The MTT assay revealed that the absorbance (A) of CBP gene transfected cells (0.2787±0.0786) had less proliferation than control cells (0.5089±0.1301) and pcDNA3.0(-) transfected cells (0.4804±0. 1547, P<0.01).Bodyen chamber assay indicated the number of migrated CBP gene transfected cells was (3.52±0.77),significantly less than in control cells (8.17±0.86) and pcDNA3.0 (-)transfected cells(8.22±1.30, P<0.01). Conclusion The CBP gene,as a recently identified transmembrane protein, can restrain malignant phenotypes of the human lung adencarcinoma in vitro and may participate in construction of neg-ative feedback loop of SFKs to inhibit the growth, migration and invasion of lung cancer cells.