中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
3期
222-227
,共6页
王川%亢渝俊%姜政%王丕龙
王川%亢渝俊%薑政%王丕龍
왕천%항투준%강정%왕비룡
水甘油通道蛋白质类%RNA干扰%基因沉默%短发夹RNA%脂肪肝,非酒精性
水甘油通道蛋白質類%RNA榦擾%基因沉默%短髮夾RNA%脂肪肝,非酒精性
수감유통도단백질류%RNA간우%기인침묵%단발협RNA%지방간,비주정성
Aquaglyceroporins%RNA interference%Gene silencing%Short hairpin RNA%Fatty liver,nonalcoholic
目的 构建人水甘油通道蛋白9 (AQP9)短发夹RNA (shRNA),筛选出沉默效果明显的重组质粒,检测AQP9的sh.RNA对非酒精性脂肪性肝病(NAFLD)细胞模型的作用.方法 设计合成针对人AQP9的小干扰RNA,退火形成双链shRNA后插入pGenesil-1质粒中,并将其转染L02肝细胞,逆转录-聚合酶链反应(RT-PCR)及Western blot筛选出沉默效果明显的重组质粒.经油酸诱导L02细胞脂肪变性建立NAFLD细胞模型,通过油红O染色,测定甘油三酯、游离脂肪酸及甘油含量,检测重组质粒对NAFLD细胞模型的作用.数据比较采用方差分析.结果 经RTPCR及Western blot筛选,pshRNA-AQP9a转染组mRNA及蛋白质相对表达率为25.10％±1.19％及25.41％±1.96％,与未处理L02细胞组的39.33％±1.69％及35.08％±1.89％相比,均明显降低(P值均＜ 0.01);将重组质粒pshRNA-AQP9a转染NAFLD细胞模型能够降低其AQP9的mRNA及蛋白质表达量;油红O染色结果显示,油酸/pshRNA-AQP9a转染组细胞内脂质含量明显减少.油酸/pshRNA-AQP9a转染组细胞内甘油三酯、游离脂肪酸及甘油含量分别为(2.738±0.231) mmol/L、(1.182±0.168)mmol/L和(0.730±0.084) mmol/L,油酸组分别为(3.218±0.220) mmol/L、(1.538±0.193)mmol/L及(1.024±0.148) mmol/L,油酸/pshRNA-AQP9a转染组均明显降低(P值均＜0.05).结论 降低AQP9表达量能够减轻NAFLD细胞模型的脂肪变性程度,为进一步研究AQP9对NAFLD的基因治疗奠定基础.
目的 構建人水甘油通道蛋白9 (AQP9)短髮夾RNA (shRNA),篩選齣沉默效果明顯的重組質粒,檢測AQP9的sh.RNA對非酒精性脂肪性肝病(NAFLD)細胞模型的作用.方法 設計閤成針對人AQP9的小榦擾RNA,退火形成雙鏈shRNA後插入pGenesil-1質粒中,併將其轉染L02肝細胞,逆轉錄-聚閤酶鏈反應(RT-PCR)及Western blot篩選齣沉默效果明顯的重組質粒.經油痠誘導L02細胞脂肪變性建立NAFLD細胞模型,通過油紅O染色,測定甘油三酯、遊離脂肪痠及甘油含量,檢測重組質粒對NAFLD細胞模型的作用.數據比較採用方差分析.結果 經RTPCR及Western blot篩選,pshRNA-AQP9a轉染組mRNA及蛋白質相對錶達率為25.10％±1.19％及25.41％±1.96％,與未處理L02細胞組的39.33％±1.69％及35.08％±1.89％相比,均明顯降低(P值均＜ 0.01);將重組質粒pshRNA-AQP9a轉染NAFLD細胞模型能夠降低其AQP9的mRNA及蛋白質錶達量;油紅O染色結果顯示,油痠/pshRNA-AQP9a轉染組細胞內脂質含量明顯減少.油痠/pshRNA-AQP9a轉染組細胞內甘油三酯、遊離脂肪痠及甘油含量分彆為(2.738±0.231) mmol/L、(1.182±0.168)mmol/L和(0.730±0.084) mmol/L,油痠組分彆為(3.218±0.220) mmol/L、(1.538±0.193)mmol/L及(1.024±0.148) mmol/L,油痠/pshRNA-AQP9a轉染組均明顯降低(P值均＜0.05).結論 降低AQP9錶達量能夠減輕NAFLD細胞模型的脂肪變性程度,為進一步研究AQP9對NAFLD的基因治療奠定基礎.
목적 구건인수감유통도단백9 (AQP9)단발협RNA (shRNA),사선출침묵효과명현적중조질립,검측AQP9적sh.RNA대비주정성지방성간병(NAFLD)세포모형적작용.방법 설계합성침대인AQP9적소간우RNA,퇴화형성쌍련shRNA후삽입pGenesil-1질립중,병장기전염L02간세포,역전록-취합매련반응(RT-PCR)급Western blot사선출침묵효과명현적중조질립.경유산유도L02세포지방변성건립NAFLD세포모형,통과유홍O염색,측정감유삼지、유리지방산급감유함량,검측중조질립대NAFLD세포모형적작용.수거비교채용방차분석.결과 경RTPCR급Western blot사선,pshRNA-AQP9a전염조mRNA급단백질상대표체솔위25.10％±1.19％급25.41％±1.96％,여미처리L02세포조적39.33％±1.69％급35.08％±1.89％상비,균명현강저(P치균＜ 0.01);장중조질립pshRNA-AQP9a전염NAFLD세포모형능구강저기AQP9적mRNA급단백질표체량;유홍O염색결과현시,유산/pshRNA-AQP9a전염조세포내지질함량명현감소.유산/pshRNA-AQP9a전염조세포내감유삼지、유리지방산급감유함량분별위(2.738±0.231) mmol/L、(1.182±0.168)mmol/L화(0.730±0.084) mmol/L,유산조분별위(3.218±0.220) mmol/L、(1.538±0.193)mmol/L급(1.024±0.148) mmol/L,유산/pshRNA-AQP9a전염조균명현강저(P치균＜0.05).결론 강저AQP9표체량능구감경NAFLD세포모형적지방변성정도,위진일보연구AQP9대NAFLD적기인치료전정기출.
Objective To construct a short hairpin (sh)RNA targeting aquaglyceroporin 9 (AQP9) that effectively silences gene expression in liver cells in order to investigate of the role of AQP9 in nonalcoholic fatty liver disease (NAFLD) pathogenesis using an in vitro cell model system.Methods Small interfering (si)RNAs were designed against the human gene sequences encoding AQP9 (NCBI GenBank Accession No.AB008775) and unrelated control sequences,synthesized,annealed to form double-strands,and inserted into the pGenesil-1 shRNA-expression plasmid.The silencing effects of the four pshRNA-AQP9 constructs (a-d) and the pshRNA-negative control construct were investigated by transfecting into the L02 human normal liver cell line and detecting expression ofAQP9 mRNA and protein (relative to β-actin) by reverse transcription-PCR and western blotting.The NAFLD cell model was established by treating L02 cells with oleic acid to induce fatty degeneration.After transfecting the NAFLD cell model with various constructs,the effects on NAFLD-related features were investigated by staining with Oil Red O (to detect lipid droplets) and performing enzymatic assays (to quantitate triglyceride (TG),free fatty acid (FFA) and glycerol content).The significance of intergroup differences was assessed by analysis of variance test.Results Ofthe four pshRNAAQP9 constructs,pshRNA-AQP9a produced the most robust silencing effect on AQP9 mRNA (25.1 ± 1.2％ vs.untransfected:39.3 ± 1.7％ and pshRNA-negative control:39.4 ± 1.5％,P ＜ 0.01) and protein (25.4 ± 2.0％ vs.untransfected:35.1 ± 1.9％ and psh-RNA-negative control:35.6 ± 2.3％,P ＜ 0.01).Oleic acid-induced L02 cells showed enhanced AQP9 mRNA and protein expression,and increased intracellular content of lipid,TG,FFA,and glycerol,which were significantly reduced by pshRNA-AQP9a transfection (allP ＜ 0.05).Conclusion The new pshRNA-AQP9a construct can efficiently reduce AQP9 expression in cultured human liver cells and relieve steatosis-related features in an NAFLD cell model.pshRNA-AQP9a represents a novel tool for studying the role of AQP9 in NAFLD pathogenesis and its potential as a gene therapy strategy.
