山西医科大学学报
山西醫科大學學報
산서의과대학학보
JOURNAL OF SHANXI MEDICAL UNIVERSITY
2015年
1期
1119-1122,1123
,共5页
李菁华%刘震雄%赵曙光%张哲%闻勤生%王景杰%张明鑫
李菁華%劉震雄%趙曙光%張哲%聞勤生%王景傑%張明鑫
리정화%류진웅%조서광%장철%문근생%왕경걸%장명흠
Nrf2%短发夹状RNA%慢病毒载体%HSC-T6
Nrf2%短髮夾狀RNA%慢病毒載體%HSC-T6
Nrf2%단발협상RNA%만병독재체%HSC-T6
Nrf2%short hairpin RNA%lentiviral vector%HSC-T6 cells
目的：设计以NF-E2-related factor2(Nrf2)基因为靶点的短发夹状RNA(shRNA),构建重组慢病毒表达载体并转染大鼠肝星状细胞株HSC-T6,观察其对Nrf2表达的影响。方法应用重组DNA技术,将设计好的4条基因特异性shRNA序列插至慢病毒表达载体pGLV3-GFP中,构建pGLV3-GFP-Nrf2-shRNA-1/2/3/4,并应用脂质体法转染293T细胞,进行病毒包装及滴度测定。采用实时荧光定量PCR( RT-PCR)及Western blot分别从mRNA和蛋白水平检测转染72 h和96 h后大鼠肝星状细胞株HSC-T6 Nrf2基因的表达情况。结果测序证实慢病毒载体构建成功,并测定滴度为1×109 TU/ml,pGLV3-GFP-Nrf2-shRNA转染后72 h和96 h可抑制Nrf2基因mRNA及蛋白的表达。结论成功构建Nrf2基因的shRNA慢病毒载体,可有效抑制大鼠肝星状细胞株HSC-T6中Nrf2基因的表达。
目的：設計以NF-E2-related factor2(Nrf2)基因為靶點的短髮夾狀RNA(shRNA),構建重組慢病毒錶達載體併轉染大鼠肝星狀細胞株HSC-T6,觀察其對Nrf2錶達的影響。方法應用重組DNA技術,將設計好的4條基因特異性shRNA序列插至慢病毒錶達載體pGLV3-GFP中,構建pGLV3-GFP-Nrf2-shRNA-1/2/3/4,併應用脂質體法轉染293T細胞,進行病毒包裝及滴度測定。採用實時熒光定量PCR( RT-PCR)及Western blot分彆從mRNA和蛋白水平檢測轉染72 h和96 h後大鼠肝星狀細胞株HSC-T6 Nrf2基因的錶達情況。結果測序證實慢病毒載體構建成功,併測定滴度為1×109 TU/ml,pGLV3-GFP-Nrf2-shRNA轉染後72 h和96 h可抑製Nrf2基因mRNA及蛋白的錶達。結論成功構建Nrf2基因的shRNA慢病毒載體,可有效抑製大鼠肝星狀細胞株HSC-T6中Nrf2基因的錶達。
목적：설계이NF-E2-related factor2(Nrf2)기인위파점적단발협상RNA(shRNA),구건중조만병독표체재체병전염대서간성상세포주HSC-T6,관찰기대Nrf2표체적영향。방법응용중조DNA기술,장설계호적4조기인특이성shRNA서렬삽지만병독표체재체pGLV3-GFP중,구건pGLV3-GFP-Nrf2-shRNA-1/2/3/4,병응용지질체법전염293T세포,진행병독포장급적도측정。채용실시형광정량PCR( RT-PCR)급Western blot분별종mRNA화단백수평검측전염72 h화96 h후대서간성상세포주HSC-T6 Nrf2기인적표체정황。결과측서증실만병독재체구건성공,병측정적도위1×109 TU/ml,pGLV3-GFP-Nrf2-shRNA전염후72 h화96 h가억제Nrf2기인mRNA급단백적표체。결론성공구건Nrf2기인적shRNA만병독재체,가유효억제대서간성상세포주HSC-T6중Nrf2기인적표체。
Objective To construct lentiviral vector targeting NF-E2-related factor2 ( Nrf2 ) gene and evaluate its silencing effect on Nrf2 gene in rat hepatic stellate cell line HSC-T6. Methods Four short hairpin RNA( shRNA) fragments targeting Nrf2 were de-signed and cloned into lentiviral vector pGLV3-GFP to construct pGLV3-GFP-Nrf2-shRNA1/2/3/4. Then the silencing effects on Nrf2 gene were confirmed by real-time PCR and Western blot in transfected HSC-T6 cells at 72 h and 96 h. Results The Nrf2 shRNA lentiviral vectors were successfully constructed and confirmed by sequencing when the virus reached a titer of 1 × 109 TU/ml. The expres-sion of Nrf2 in both mRNA and protein levels were inhibited by real-time PCR and Western blot. Conclusion The shRNA expressing lentiviral recombinants targeting the Nrf2 gene are successfully constructed and could effectively silence expression of Nrf2 gene in HSC-T6 cells.
