CAJ | 학술논문

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.
목적 구건p110β기인고표체질립,전염위암MKN28세포,관찰기대위암MKN28세포증식적영향.방법 이기인조cDNA위모판,취합매련반응(PCR)확증목적편단,구건pCMV-Flag-p110β질립,통과매절감정급측서학정무오후,전염MKN28세포,통과실시정량PCR(Real-timePCR)화Western blot검측p110β고표체,이급통과새서람(MTT)법검측기대MKN28세포증식적영향.결과 성공구건료Flag-p110β질립,재위암MKN28세포중고표체p110β능촉진단백격매B(AKT)적린산화,48 h후새서람(MTT)법검측공백조、음성대조조화고표체p110PIK조490 nm파장처적흡광도치분별위:0.791 6±0.0425、0.828 1 ±0.015 6、1.031 2±0.1094,차이유통계학의의(P＜0.05).결론 성공구건Flag-p110β질립,고표체p110β능구촉진MKN28세포증식.
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.