中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2013年
3期
213-217
,共5页
赵燕颖%李亚刚%孙远杰%刘海鹏%杨泽成%张舵舵%赵春燕
趙燕穎%李亞剛%孫遠傑%劉海鵬%楊澤成%張舵舵%趙春燕
조연영%리아강%손원걸%류해붕%양택성%장타타%조춘연
癌,肝细胞%基因沉默%RNA,小分子干扰%基因,p53%鼠双微粒体-2基因
癌,肝細胞%基因沉默%RNA,小分子榦擾%基因,p53%鼠雙微粒體-2基因
암,간세포%기인침묵%RNA,소분자간우%기인,p53%서쌍미립체-2기인
Carcinoma,hepatocellular%Gene silencing%RNA,small interfering%Genes,p53%Murine double minute 2
目的 构建鼠双微粒体-2基因(MDM2)靶向小分子干扰RNA (siRNA)质粒,研究其对人肝癌HepG2细胞裸鼠皮下移植瘤生长的抑制作用,探讨其在肝癌基因治疗中的可行性.方法 构建MDM2的siRNA真核表达载体pSilencer-siRNA-MDM2 (siMDM2),建立裸鼠荷瘤模型,分别给予阴性对照质粒和siMDM2质粒处理.监测肿瘤生长变化,RT-PCR、Western blot方法检测MDM2及p53 mRNA和蛋白的表达变化.正态分布数据的组间比较用单因素方差分析及LSD检验.结果 裸鼠体内实验结果显示,转染siMDM2-1和siMDM2-2组与对照组相比,肿瘤增长受到明显抑制,抑瘤率分别为60.6％和54.6％.RT-PCR和Westem blot结果示MDM2的mRNA和蛋白质表达下调,而p53的mRNA和蛋白质表达上调.与空白组比较,转染siMDM2-1和siMDM2-2组的MDM2mRNA表达分别下调62.8％和61.5％,MDM2蛋白表达分别下调60.7％和59.5％;p53 mRNA表达分别上调47.1％和45.6％,p53蛋白表达分别上调45.9％和44.3％,差异均具有统计学意义(F值分别为75.099、47.860、17.676和235.770,P值均＜0.01).结论 siRNA沉默MDM2基因能抑制人肝癌HepG2细胞裸鼠皮下移植瘤的形成,可能与其在体内有效下调MDM2和上调p53的mRNA及蛋白质表达有关.
目的 構建鼠雙微粒體-2基因(MDM2)靶嚮小分子榦擾RNA (siRNA)質粒,研究其對人肝癌HepG2細胞裸鼠皮下移植瘤生長的抑製作用,探討其在肝癌基因治療中的可行性.方法 構建MDM2的siRNA真覈錶達載體pSilencer-siRNA-MDM2 (siMDM2),建立裸鼠荷瘤模型,分彆給予陰性對照質粒和siMDM2質粒處理.鑑測腫瘤生長變化,RT-PCR、Western blot方法檢測MDM2及p53 mRNA和蛋白的錶達變化.正態分佈數據的組間比較用單因素方差分析及LSD檢驗.結果 裸鼠體內實驗結果顯示,轉染siMDM2-1和siMDM2-2組與對照組相比,腫瘤增長受到明顯抑製,抑瘤率分彆為60.6％和54.6％.RT-PCR和Westem blot結果示MDM2的mRNA和蛋白質錶達下調,而p53的mRNA和蛋白質錶達上調.與空白組比較,轉染siMDM2-1和siMDM2-2組的MDM2mRNA錶達分彆下調62.8％和61.5％,MDM2蛋白錶達分彆下調60.7％和59.5％;p53 mRNA錶達分彆上調47.1％和45.6％,p53蛋白錶達分彆上調45.9％和44.3％,差異均具有統計學意義(F值分彆為75.099、47.860、17.676和235.770,P值均＜0.01).結論 siRNA沉默MDM2基因能抑製人肝癌HepG2細胞裸鼠皮下移植瘤的形成,可能與其在體內有效下調MDM2和上調p53的mRNA及蛋白質錶達有關.
목적 구건서쌍미립체-2기인(MDM2)파향소분자간우RNA (siRNA)질립,연구기대인간암HepG2세포라서피하이식류생장적억제작용,탐토기재간암기인치료중적가행성.방법 구건MDM2적siRNA진핵표체재체pSilencer-siRNA-MDM2 (siMDM2),건립라서하류모형,분별급여음성대조질립화siMDM2질립처리.감측종류생장변화,RT-PCR、Western blot방법검측MDM2급p53 mRNA화단백적표체변화.정태분포수거적조간비교용단인소방차분석급LSD검험.결과 라서체내실험결과현시,전염siMDM2-1화siMDM2-2조여대조조상비,종류증장수도명현억제,억류솔분별위60.6％화54.6％.RT-PCR화Westem blot결과시MDM2적mRNA화단백질표체하조,이p53적mRNA화단백질표체상조.여공백조비교,전염siMDM2-1화siMDM2-2조적MDM2mRNA표체분별하조62.8％화61.5％,MDM2단백표체분별하조60.7％화59.5％;p53 mRNA표체분별상조47.1％화45.6％,p53단백표체분별상조45.9％화44.3％,차이균구유통계학의의(F치분별위75.099、47.860、17.676화235.770,P치균＜0.01).결론 siRNA침묵MDM2기인능억제인간암HepG2세포라서피하이식류적형성,가능여기재체내유효하조MDM2화상조p53적mRNA급단백질표체유관.
Objecive To construct a short hairpin (sh)RNA targeting the gene encoding the MDM2 oncoprotein in order to investigate its role in human hepatocellular carcinoma (HCC) and its potential for use as a gene therapy strategy to inhibit HCC growth in vivo.Methods Small interfering (si)RNAs were designed targeting the MDM2 gene (siMDM2-1 and siMDM2-2) and unrelated sequences (negative control) and cloned into the expression plasmid pGCSilencer-U6-neo-GFP.A HCC mouse model was established by subcutaneous inoculation of HepG2 cells (2 x 106 in 0.2 ml) into 20 nude mice.The inoculated mice were divided into four equal groups for tumor-localized injections of saline,negative control siRNA plasmid,siMDM2-1 plasmid,and siMDM2-2 plasmid.Tumor growth was observed daily (by caliper measurement) for one month,when mice were sacrificed by cervical dislocation.The tumor mass was resected for analysis of tumor inhibition rate (％ =[(average tumor weight of control group-average tumor weight of treatment group) / average tumor weight of control group x 100]) and effects on MDM2 and p53 mRNA and protein expression (by reverse transcription PCR and western blotting,both normalized to β-actin).Significance of between-group differences was assessed by one-way ANOVA or LSD test；pairwise comparisons were made by the Chi-squared test.Results Both siMDM2-1 and siMDM2-2 suppressed the xenografted tumor growth remarkably (60.6％ and 54.6％ inhibition rates,respectively),significantly reduced the expression of MDM2 gene (62.8％ and 61.6％) and protein (60.7％ and 59.5％),and significantly increased p53 gene (47.1％ and 45.6％) and protein (45.9％ and 44.3％) (all,P ＜0.05).Conclusion shRNA-mediated silencing of the MDM2 gene effectively inhibits HCC tumorigenesis of subcutaneously xenografted HepG2 cells in nude mice,and the mechanism may involve p53.
