中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2011年
35期
2497-2500
,共4页
周奇%周祥兵%王亚峰%梁力建%彭宝岗
週奇%週祥兵%王亞峰%樑力建%彭寶崗
주기%주상병%왕아봉%량력건%팽보강
肝肿瘤%凝血致活酶%RNA干扰%肿瘤转移
肝腫瘤%凝血緻活酶%RNA榦擾%腫瘤轉移
간종류%응혈치활매%RNA간우%종류전이
Liver neoplasms%Thromboplastin%RNA interference%Neoplasm metastasis
目的 应用RNA干扰(RNAi)技术抑制人类肝癌细胞内组织因子(TF)基因表达，从而探讨对其侵袭转移能力的影响。方法 本实验以人类肝癌细胞系HepG2细胞株为实验对象，将携带有针对TF小干扰RNA( siRNA)序列的质粒pGPU6/GFP/Neo-TF siRNA，转染HepG2细胞株，以适当浓度的G418筛选获得阳性克隆，通过RT-PCR和蛋白质印迹法比较转染TF siRNA(+)质粒、转染TF siRNA( -)质粒以及未转染任何质粒的HepG2细胞内源性TF在基因及蛋白质表达水平的差异，进而应用Transwell体外侵袭实验检测三者的侵袭转移能力，探讨TF对肿瘤细胞侵袭转移能力的影响。结果 (1) RT-PCR结果 显示，转染TF siRNA(+)质粒的HepG2细胞其内源性TF表达水平低于转染TF siRNA（-）质粒和未转染质粒的表达水平。(2)蛋白质Western印迹法结果 显示，转染TFsiRNA(+)质粒的HepG2细胞其内源性TF蛋白表达水平低于转染TF siRNA( -)质粒和未转染质粒的表达水平。(3) Transwell体外侵袭实验结果 显示，转染TF siRNA(+)质粒的HepG2穿膜细胞数[(14±10)个]少于转染TF siRNA（-）质粒的HepG2穿膜细胞数[(128±18)个]，经t检验比较，两组差异有统计学意义（P＜0.05）。这提示应用RNA干扰技术抑制肿瘤细胞TF表达可以使HepG2细胞侵袭转移能力显著下降。结论 TF与肝癌细胞的侵袭转移能力密切相关，应用RNA干扰技术抑制其表达可以明显降低细胞HepG2体外侵袭转移能力。
目的 應用RNA榦擾(RNAi)技術抑製人類肝癌細胞內組織因子(TF)基因錶達，從而探討對其侵襲轉移能力的影響。方法 本實驗以人類肝癌細胞繫HepG2細胞株為實驗對象，將攜帶有針對TF小榦擾RNA( siRNA)序列的質粒pGPU6/GFP/Neo-TF siRNA，轉染HepG2細胞株，以適噹濃度的G418篩選穫得暘性剋隆，通過RT-PCR和蛋白質印跡法比較轉染TF siRNA(+)質粒、轉染TF siRNA( -)質粒以及未轉染任何質粒的HepG2細胞內源性TF在基因及蛋白質錶達水平的差異，進而應用Transwell體外侵襲實驗檢測三者的侵襲轉移能力，探討TF對腫瘤細胞侵襲轉移能力的影響。結果 (1) RT-PCR結果 顯示，轉染TF siRNA(+)質粒的HepG2細胞其內源性TF錶達水平低于轉染TF siRNA（-）質粒和未轉染質粒的錶達水平。(2)蛋白質Western印跡法結果 顯示，轉染TFsiRNA(+)質粒的HepG2細胞其內源性TF蛋白錶達水平低于轉染TF siRNA( -)質粒和未轉染質粒的錶達水平。(3) Transwell體外侵襲實驗結果 顯示，轉染TF siRNA(+)質粒的HepG2穿膜細胞數[(14±10)箇]少于轉染TF siRNA（-）質粒的HepG2穿膜細胞數[(128±18)箇]，經t檢驗比較，兩組差異有統計學意義（P＜0.05）。這提示應用RNA榦擾技術抑製腫瘤細胞TF錶達可以使HepG2細胞侵襲轉移能力顯著下降。結論 TF與肝癌細胞的侵襲轉移能力密切相關，應用RNA榦擾技術抑製其錶達可以明顯降低細胞HepG2體外侵襲轉移能力。
목적 응용RNA간우(RNAi)기술억제인류간암세포내조직인자(TF)기인표체，종이탐토대기침습전이능력적영향。방법 본실험이인류간암세포계HepG2세포주위실험대상，장휴대유침대TF소간우RNA( siRNA)서렬적질립pGPU6/GFP/Neo-TF siRNA，전염HepG2세포주，이괄당농도적G418사선획득양성극륭，통과RT-PCR화단백질인적법비교전염TF siRNA(+)질립、전염TF siRNA( -)질립이급미전염임하질립적HepG2세포내원성TF재기인급단백질표체수평적차이，진이응용Transwell체외침습실험검측삼자적침습전이능력，탐토TF대종류세포침습전이능력적영향。결과 (1) RT-PCR결과 현시，전염TF siRNA(+)질립적HepG2세포기내원성TF표체수평저우전염TF siRNA（-）질립화미전염질립적표체수평。(2)단백질Western인적법결과 현시，전염TFsiRNA(+)질립적HepG2세포기내원성TF단백표체수평저우전염TF siRNA( -)질립화미전염질립적표체수평。(3) Transwell체외침습실험결과 현시，전염TF siRNA(+)질립적HepG2천막세포수[(14±10)개]소우전염TF siRNA（-）질립적HepG2천막세포수[(128±18)개]，경t검험비교，량조차이유통계학의의（P＜0.05）。저제시응용RNA간우기술억제종류세포TF표체가이사HepG2세포침습전이능력현저하강。결론 TF여간암세포적침습전이능력밀절상관，응용RNA간우기술억제기표체가이명현강저세포HepG2체외침습전이능력。
Objective To study the effect of tissue factor (TF) on human hepatocellular carcinoma cells when their inner TF gene was knocked down by RNA interference. Methods The eukaryotic expression vector bearing siRNA sequence that targeted at TF gene was transfected into human hepatocellular carcinoma cell line HepG2. RT-PCR and Western blot were used to detect the changes of TF gene expression. Transwell invasion assay invitro were used to show the invasive ability of HepG2 cells with transfected pGPU6/GFP/Neo-TFsiRNA. Results RT-PCR and Western blot analysis showed that HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased the endogenous TF gene expression. Correspondingly the mean invading cells was 14 ± 10 for pGPU6/GFP/Neo-TFsiRNA transfection and 128 ± 18 for the NCsiRNA transfection,respectively by the Transwell invasion test in vitro( P ＜0.05 ). The results indicated that the invasive ability of the HepG2 cells with pGPU6/GFP/Neo-TFsiRNA transfected decreased obviously. Conclusions TF is involved in the progression of hepatocellular carcinoma and inhibiting the expression of TF can decrease the invasion and metastasis ability of tumor cell in vitro.
