摘要
目的 构建p110β基因高表达质粒,转染胃癌MKN28细胞,观察其对胃癌MKN28细胞增殖的影响.方法 以基因组cDNA为模板,聚合酶链反应（PCR）扩增目的片段,构建pCMV-Flag-p110β质粒,通过酶切鉴定及测序确定无误后,转染MKN28细胞,通过实时定量PCR（Real-timePCR）和Western blot检测p110β高表达,以及通过噻唑蓝（MTT）法检测其对MKN28细胞增殖的影响.结果 成功构建了Flag-p110β质粒,在胃癌MKN28细胞中高表达p110β能促进蛋白激酶B（AKT）的磷酸化,48 h后噻唑蓝（MTT）法检测空白组、阴性对照组和高表达p110PIK组490 nm波长处的吸光度值分别为：0.791 6±0.0425、0.828 1 ±0.015 6、1.031 2±0.1094,差异有统计学意义（P＜0.05）.结论 成功构建Flag-p110β质粒,高表达p110β能够促进MKN28细胞增殖.
Objective To construct the plasmid of p110β,and observe the influence of p110β on growth of gastric cancer MKN28 cells after transfected with Flag-p110β.Methods p110β cDNA was amplified by polymerase chain reaction （PCR）,with the genomic DNA by reverse transcription.The vector of pCMV-Flag-p110β was constructed,and identified by restriction endonuclease digestion and sequencing.The expression levels of p110β and p-protein kinase B （p-AKT） after transfection of the vector of Flag-p110βwere detected by using Western blotting.Methyl thiazol tetrazolium （MTT） assay was used to measure the influence of p110β on growth of gastric cancer MKN28 cells.Results The Flag-pl l0 vector was successfully constructed and over-expression of p110β could increase the p-AKT level.At 48 h after transfection,MTT assay the A490 nm in blank,control and p110β was 0.791 6 ±0.042 5,0.828 1 ±0.015 6 and 1.031 2 ±0.109 4 respectively with the difference being significant among them.Conclusion The p110β vector was successfully constructed and over-expression of p110β could promote growth of MKN28 cells.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2014年第3期477-479,共3页
Chinese Journal of Experimental Surgery
基金
国家973计划资助项目（2009CB521802）
国家自然科学基金资助项目（30872472、30973496、30800569）
关键词
p110β
胃癌
细胞增殖
p110β
Gastric carcinoma
Cell proliferation
