中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2010年
8期
609-613
,共5页
王以浪%姚登福%吴玮%赛文莉%邱历伟%杨君伶%朱建伟
王以浪%姚登福%吳瑋%賽文莉%邱歷偉%楊君伶%硃建偉
왕이랑%요등복%오위%새문리%구력위%양군령%주건위
癌,肝细胞%NF-κB%细胞凋亡%小干扰RNA
癌,肝細胞%NF-κB%細胞凋亡%小榦擾RNA
암,간세포%NF-κB%세포조망%소간우RNA
Carcinoma,hepatocellular%NF-kappa B%Apoptosis%Small interference RNA
目的 探讨小干扰RNA(SiRNA)抑制核转录因子(NF)-κB活化对肝癌细胞凋亡的影响.方法 化学合成NF-κB siRNA和阴性对照siRNA,脂质体法转染HepG2细胞,用巢式RT-PCR和荧光定量PCR检测NF-κB mRNA表达情况;免疫组织化学法、酶联免疫吸附法、Western b10t检测NF-κB蛋白表达情况;用磷脂结合蛋白V-异硫氰酸荧光素法检测细胞凋亡,分析NF-κB表达抑制和细胞凋亡间关系.多个样本均数间的比较先行方差齐性检验,方差齐时行单因素方差分析;计数资料比较采用确切概率法分析.结果 NF-κBp65 mRNA在HepG2细胞相对表达量为1.13±0.03,在正常肝细胞L02为0.29±0.07,两者比较,t=27.02,P<0.05,差异有统计学意义.利用NF-κB siRNA干扰可下调NF-κB表达,且呈剂量、时间依赖;NF-κB siRNA转染HepG2细胞72h后,NF-κBmRNA和蛋白表达分别下降了93%和62%,抑制NF-κB表达使HepG2细胞凋亡增加85%. 结论 NF-κB在肝癌细胞中高表达,NF-κB SiRNA能特异性抑制其在肝癌细胞中活化并促进癌细胞凋亡发生.
目的 探討小榦擾RNA(SiRNA)抑製覈轉錄因子(NF)-κB活化對肝癌細胞凋亡的影響.方法 化學閤成NF-κB siRNA和陰性對照siRNA,脂質體法轉染HepG2細胞,用巢式RT-PCR和熒光定量PCR檢測NF-κB mRNA錶達情況;免疫組織化學法、酶聯免疫吸附法、Western b10t檢測NF-κB蛋白錶達情況;用燐脂結閤蛋白V-異硫氰痠熒光素法檢測細胞凋亡,分析NF-κB錶達抑製和細胞凋亡間關繫.多箇樣本均數間的比較先行方差齊性檢驗,方差齊時行單因素方差分析;計數資料比較採用確切概率法分析.結果 NF-κBp65 mRNA在HepG2細胞相對錶達量為1.13±0.03,在正常肝細胞L02為0.29±0.07,兩者比較,t=27.02,P<0.05,差異有統計學意義.利用NF-κB siRNA榦擾可下調NF-κB錶達,且呈劑量、時間依賴;NF-κB siRNA轉染HepG2細胞72h後,NF-κBmRNA和蛋白錶達分彆下降瞭93%和62%,抑製NF-κB錶達使HepG2細胞凋亡增加85%. 結論 NF-κB在肝癌細胞中高錶達,NF-κB SiRNA能特異性抑製其在肝癌細胞中活化併促進癌細胞凋亡髮生.
목적 탐토소간우RNA(SiRNA)억제핵전록인자(NF)-κB활화대간암세포조망적영향.방법 화학합성NF-κB siRNA화음성대조siRNA,지질체법전염HepG2세포,용소식RT-PCR화형광정량PCR검측NF-κB mRNA표체정황;면역조직화학법、매련면역흡부법、Western b10t검측NF-κB단백표체정황;용린지결합단백V-이류청산형광소법검측세포조망,분석NF-κB표체억제화세포조망간관계.다개양본균수간적비교선행방차제성검험,방차제시행단인소방차분석;계수자료비교채용학절개솔법분석.결과 NF-κBp65 mRNA재HepG2세포상대표체량위1.13±0.03,재정상간세포L02위0.29±0.07,량자비교,t=27.02,P<0.05,차이유통계학의의.이용NF-κB siRNA간우가하조NF-κB표체,차정제량、시간의뢰;NF-κB siRNA전염HepG2세포72h후,NF-κBmRNA화단백표체분별하강료93%화62%,억제NF-κB표체사HepG2세포조망증가85%. 결론 NF-κB재간암세포중고표체,NF-κB SiRNA능특이성억제기재간암세포중활화병촉진암세포조망발생.
Objective To investigate the effect of siRNA-mediated inhibition ofNF-κB on apoptosis of hepatocarcinoma cells. Methods Specific small interfering RNA Targeting NF-κB gene was synthesized and transfected into HepG2 cells by liposomes. Nested RT-PCR and quantitative RT-PCR were used to dectect the mRNA expression of NF-κB. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blot were performed to examine the protein expression of NF-κB.Annexin V-FITC was used to test cell apoptosis. Results The expressin of NF-κB in HepG2 cells (1.13±0.03)was significantly higher(t=27.02, P<0.05) than that in normal hepatocytes (0.29±0.07).The down-regulation of NF-κB expression was depended on the dosage of siRNA and the time after transfection. 72 h after siRNA transfection,NF-κB expression reduced by 93% and 62% at the mRNA and protein levels, respectively. The apoptosis of HepG2 cells increased by 85% with NF-κB inhibition. Conclusions NF-κB is abnomally active in HepG2 cells and NF-κB inhibition mediated by siRNA promotes HepG2 cells apoptosis. It suggested that NF-κB could be a potential target for HCC prevention gene therapy.
