CAJ | 학술논문

目的探讨慢病毒表达载体介导HIF-1α RNA干扰(RNAi)对人胰腺癌细胞Patu8988HIF-1α和Glut-1表达的影响.方法构建针对HIF-1α基因的RNAi慢病毒表达载体LV-RNAi-HIF-1α.乏氧条件下体外培养4h,转染LV-RNAi-HIF-1α的Patu8988细胞为实验组；转染空病毒载体和未转染任何病毒载体Patu8988细胞分别为阴性对照组和空白对照组.采用荧光定量RT-PCR和Western印迹法检测Patu8988细胞HIF-1α表达情况,采用RT-PCR法检测转染LV-RNAi-HIF-1α后Patu8988细胞Glut-1的表达情况.采用SPSS 17.0软件行单因素方差分析和两样本t检验分析各组之间的差异.结果构建的慢病毒表达载体LV-RNAi-HIF-1α,在常氧和乏氧状态下致HIF-1α mRNA表达分别下降65.1％(0.209/0.321)和80.6％ (0.791/0.982)(t=10.52和15.24,均P＜0.05),阴性对照组分别为0.6％(0.002/0.321)和7.2％(0.071/0.982)(t=5.26和7.38,均P＜0.05)；乏氧状态下实验组、阴性对照组和空白对照组HIF-1α蛋白的表达分别为0.159±0.010、0.745±0.012和0.711±0.023,差异有统计学意义(F=35.52,t=6.72和10.56,均P＜0.05),实验组较其他2组表达下降；乏氧条件下实验组Glut-1 mRNA表达(0.040±0.003)较阴性对照组(0.054±0.003)和空白对照组(0.062±0.004)均有明显下降(F=35.28,t=5.94和8.55,均P＜0.01).结论通过构建LV-RNAi-HIF-1α慢病毒表达载体沉默HIF-1α基因表达,可降低Patu8988胰腺癌细胞Glut-1 mRNA表达.
목적 탐토만병독표체재체개도HIF-1α RNA간우(RNAi)대인이선암세포Patu8988HIF-1α화Glut-1표체적영향.방법 구건침대HIF-1α기인적RNAi만병독표체재체LV-RNAi-HIF-1α.핍양조건하체외배양4h,전염LV-RNAi-HIF-1α적Patu8988세포위실험조；전염공병독재체화미전염임하병독재체Patu8988세포분별위음성대조조화공백대조조.채용형광정량RT-PCR화Western인적법검측Patu8988세포HIF-1α표체정황,채용RT-PCR법검측전염LV-RNAi-HIF-1α후Patu8988세포Glut-1적표체정황.채용SPSS 17.0연건행단인소방차분석화량양본t검험분석각조지간적차이.결과 구건적만병독표체재체LV-RNAi-HIF-1α,재상양화핍양상태하치HIF-1α mRNA표체분별하강65.1％(0.209/0.321)화80.6％ (0.791/0.982)(t=10.52화15.24,균P＜0.05),음성대조조분별위0.6％(0.002/0.321)화7.2％(0.071/0.982)(t=5.26화7.38,균P＜0.05)；핍양상태하실험조、음성대조조화공백대조조HIF-1α단백적표체분별위0.159±0.010、0.745±0.012화0.711±0.023,차이유통계학의의(F=35.52,t=6.72화10.56,균P＜0.05),실험조교기타2조표체하강；핍양조건하실험조Glut-1 mRNA표체(0.040±0.003)교음성대조조(0.054±0.003)화공백대조조(0.062±0.004)균유명현하강(F=35.28,t=5.94화8.55,균P＜0.01).결론 통과구건LV-RNAi-HIF-1α만병독표체재체침묵HIF-1α기인표체,가강저Patu8988이선암세포Glut-1 mRNA표체.
Objective To investigate the inhibitory effect of the lentiviral vector (LV)-mediated RNA interference (RNAi) targeting HIF-1α on the expression of HIF-1α and Glut-1 in human pancreatic cancer Patu8988 cells.Methods The RNAi targeting HIF-1α was combined to LV,and transfected into Patu8988 cells.The Patu8988 cells transfected with the empty vector and exposed to 0.5％ O2 for 4 h served as hypoxia negative control,the Patu8988 cells not transfected with vector and exposed to 0.5％ O2 for 4 h as hypoxia blank control,and the Patu8988 cells transfected with LV-RNAi-HIF-1α and exposed to 0.5％O2 for 4 h as experimental group.The expression of HIF-1α was measured by RT-PCR and Western blot respectively.The expression of Glut-1 was measured by RT-PCR.Each group was compared according to oneway analysis of variance and two-sample t test.Results After transfection with LV-RNAi-HIF-1α,HIF-1α mRNA expression decreased by 65.1％ (0.209/0.321) and 80.6％ (0.791/0.982) (t=10.52,15.24,both P＜0.05) under normoxia and hypoxia conditions,meanwhile with the empty vector,HIF-1α mRNA expression decreased by 0.6％ (0.002/0.321) and 7.2％ (0.071/0.982) (t =5.26,7.38,both P＜0.05).Under hypoxia conditions,the protein of HIF-1α in experimental group cells (0.159±0.010) was down-regulated obviously compared to the negative control group (0.745± 0.012) and the blank control group (0.711 ± 0.023)(F=35.52,t =6.72,10.56,all P＜0.05).The expression of Glut-1 mRNA in experimental group cells (0.040±0.003) decreased obviously compared to the negative control group (0.054±0.003) and blank control group (0.062±0.004) (F=35.28,t=5.94,8.55,all P＜0.01).Conclusion Gene silencing of HIF-1α using LV-mediated RNAi can inhibit the expression of HIF-1α and decrease the expression of Glut-1mRNA in Patu8988 cells.