中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
11期
2480-2482
,共3页
郭涛%顾春东%舒鑫%郑勋%赵士磊%李锦绣
郭濤%顧春東%舒鑫%鄭勛%趙士磊%李錦繡
곽도%고춘동%서흠%정훈%조사뢰%리금수
肺腺癌%Y盒结合蛋白-1%短发夹RNA%绿色荧光蛋白
肺腺癌%Y盒結閤蛋白-1%短髮夾RNA%綠色熒光蛋白
폐선암%Y합결합단백-1%단발협RNA%록색형광단백
Lung adenocarcinoma%Y-box binding protein-1%Short hairpin RNA%Green fluorescent protein
目的 构建稳定沉默Y盒结合蛋白1(YB-1)基因的人肺腺癌A549细胞株并观察其增殖能力.方法 以脂质体将两种沉默YB-1基因的shRNA真核表达载体转染人肺腺癌细胞株A549,G418筛选稳定转染细胞株.逆转录-聚合酶链反应(RT-PCR)、实时荧光定量聚合酶链反应(FQ-PCR)及Westem blot法检测YB-1基因表达;噻唑蓝(MTT)法绘制细胞生长曲线.结果 稳定转染细胞株稳定表达绿色荧光蛋白(GFP);RT-PCR、FQ-PCR和Western blot法检测结果显示稳定转染组细胞株A549-746与A549-326 YB-1 mRNA和蛋白表达(0.41±0.01与0.39±0.06、0.43±0.10与0.20±0.24、0.49 ±0.06与0.24±0.02)较对照组明显下降(P＜0.05);噻唑蓝(MTT)检测稳定转染组较对照组生长速度降低(P＜0.05).结论 成功建立稳定表达GFP,并稳定沉默YB-1基因的人肺腺癌细胞株A549-746和A549-326,沉默YB-1可抑制A549细胞增殖能力.
目的 構建穩定沉默Y盒結閤蛋白1(YB-1)基因的人肺腺癌A549細胞株併觀察其增殖能力.方法 以脂質體將兩種沉默YB-1基因的shRNA真覈錶達載體轉染人肺腺癌細胞株A549,G418篩選穩定轉染細胞株.逆轉錄-聚閤酶鏈反應(RT-PCR)、實時熒光定量聚閤酶鏈反應(FQ-PCR)及Westem blot法檢測YB-1基因錶達;噻唑藍(MTT)法繪製細胞生長麯線.結果 穩定轉染細胞株穩定錶達綠色熒光蛋白(GFP);RT-PCR、FQ-PCR和Western blot法檢測結果顯示穩定轉染組細胞株A549-746與A549-326 YB-1 mRNA和蛋白錶達(0.41±0.01與0.39±0.06、0.43±0.10與0.20±0.24、0.49 ±0.06與0.24±0.02)較對照組明顯下降(P＜0.05);噻唑藍(MTT)檢測穩定轉染組較對照組生長速度降低(P＜0.05).結論 成功建立穩定錶達GFP,併穩定沉默YB-1基因的人肺腺癌細胞株A549-746和A549-326,沉默YB-1可抑製A549細胞增殖能力.
목적 구건은정침묵Y합결합단백1(YB-1)기인적인폐선암A549세포주병관찰기증식능력.방법 이지질체장량충침묵YB-1기인적shRNA진핵표체재체전염인폐선암세포주A549,G418사선은정전염세포주.역전록-취합매련반응(RT-PCR)、실시형광정량취합매련반응(FQ-PCR)급Westem blot법검측YB-1기인표체;새서람(MTT)법회제세포생장곡선.결과 은정전염세포주은정표체록색형광단백(GFP);RT-PCR、FQ-PCR화Western blot법검측결과현시은정전염조세포주A549-746여A549-326 YB-1 mRNA화단백표체(0.41±0.01여0.39±0.06、0.43±0.10여0.20±0.24、0.49 ±0.06여0.24±0.02)교대조조명현하강(P＜0.05);새서람(MTT)검측은정전염조교대조조생장속도강저(P＜0.05).결론 성공건립은정표체GFP,병은정침묵YB-1기인적인폐선암세포주A549-746화A549-326,침묵YB-1가억제A549세포증식능력.
Objective To establish a human lung adenocarcinoma A549 cell line with RNA interference for YB-1 gene silencing which can stably express green fluorescent protein (GFP) and to observe the effects of silencing Y-box binding protein (YB-1) gene on proliferation in lung adenocarcinoma A549 cells.Methods The human lung adenocarcinoma A549 cells were transfected with shRNA plasmid specific for the YB-1 gene,and were then selected through G418.G418-resistant clones were generated and screened using reverse transcription polymerase chain reaction (RT-PCR),real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) and Western blotting analysis.Cell proliferation was examined by methyl thiazolyl tetrazolium.Results The G418-resistant clones (A549-746 and A549-326) showed much lower level of YB-1 mRNA (0.41 ± 0.01 and 0.39 ± 0.06) than A549-shNC (P ＜ 0.05) under RT-PCR.A549-746 and A549-326 cell clones showed much lower level of YB-1 mRNA (0.43 ±0.10 and 0.20 ± 0.24) and protein expression (0.49 ± 0.06 and 0.24 ± 0.02) than A549-shNC (P ＜ 0.05) under FQ-PCR and Western blotting analysis.Meanwhile,the G418-resistan clones (A549-746 and A549-326) stably expressed GFP in vitro.The cell growth rate in YBX1-homo-746 and YBX1-homo-326 groups was decreased significantly at the first 24 h (P ＜ 0.05).Conclusion The lung cancer cell lines A549-746 and A549-326 with stable YB-1 gene silencing and GFP expression were successfully established,and silencing YB-1 gene could inhibit the proliferation of lung adenocarcinoma A549 cells.
