中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2010年
4期
300-304
,共5页
晏维%付妤%廖家智%田德安%周珍珍%王伟%张全乐%陈孝平
晏維%付妤%廖傢智%田德安%週珍珍%王偉%張全樂%陳孝平
안유%부여%료가지%전덕안%주진진%왕위%장전악%진효평
癌,肝细胞%缺氧%RNA干扰%Netrin-1%细胞骨架重组
癌,肝細胞%缺氧%RNA榦擾%Netrin-1%細胞骨架重組
암,간세포%결양%RNA간우%Netrin-1%세포골가중조
Carcinoma hepatocellular%Hypoxia%RNA interference%Netrin-1%Cytoskeletal arrangement
目的 研究缺氧对人肝癌细胞HepG2细胞骨架重组的影响及Netrin-1在细胞骨架重组中的作用.方法 通过1%O_2 低氧培养建立人肝癌细胞HepG2物理缺氧模型,观测缺氧对细胞形态的改变.构建针对Netrin-1 mRNA的干扰质粒pshRNA-Netrin-1及阴性对照质粒pGensil-1,并将其转染HepG2细胞,筛选稳定转染细胞株.观测转染pshRNA-Netrin-1质粒前后,鬼笔环肽检测缺氧对肝癌细胞对HepG2细胞骨架重组的影响,细胞迁移实验检测缺氧条件下干扰Netrin-1对细胞迁移能力的影响.同时构建Netrin-1真核表达质粒pGNET1转染HepG2细胞,观测Netrin-1对肝癌细胞骨架重组的作用.结果 低氧培养HepG2细胞胞体变得狭长,细胞连接松散.Netrin-1蛋白表达水平显著升高(P＜0.05).HepG2细胞稳定转染pshRNA-Netrin-1后,Netrin-1 mRNA表达显著下降.抑制率达73.32%±4.12%(P＜0.01).鬼笔环肽检测F-actin结果 显示,缺氧促进HepG2细胞骨架重组,转染pshRNA-Netrin-1后,缺氧促进HepG2细胞骨架重组受到显著抑制.细胞迁移实验结果 显示,缺氧条件下干扰Netrin-1的表达显著抑制HepG细胞迁移能力(P＜0.05).转染 pGNET1后,HepG2细胞骨架发生重组.结论 缺氧促进HepG2细胞骨架重组与Netrin-1表达水平升高相关,Netrin-1表达的上调可能是缺氧促进肝癌细胞骨架重组进而促进侵袭转移的机制之一.
目的 研究缺氧對人肝癌細胞HepG2細胞骨架重組的影響及Netrin-1在細胞骨架重組中的作用.方法 通過1%O_2 低氧培養建立人肝癌細胞HepG2物理缺氧模型,觀測缺氧對細胞形態的改變.構建針對Netrin-1 mRNA的榦擾質粒pshRNA-Netrin-1及陰性對照質粒pGensil-1,併將其轉染HepG2細胞,篩選穩定轉染細胞株.觀測轉染pshRNA-Netrin-1質粒前後,鬼筆環肽檢測缺氧對肝癌細胞對HepG2細胞骨架重組的影響,細胞遷移實驗檢測缺氧條件下榦擾Netrin-1對細胞遷移能力的影響.同時構建Netrin-1真覈錶達質粒pGNET1轉染HepG2細胞,觀測Netrin-1對肝癌細胞骨架重組的作用.結果 低氧培養HepG2細胞胞體變得狹長,細胞連接鬆散.Netrin-1蛋白錶達水平顯著升高(P＜0.05).HepG2細胞穩定轉染pshRNA-Netrin-1後,Netrin-1 mRNA錶達顯著下降.抑製率達73.32%±4.12%(P＜0.01).鬼筆環肽檢測F-actin結果 顯示,缺氧促進HepG2細胞骨架重組,轉染pshRNA-Netrin-1後,缺氧促進HepG2細胞骨架重組受到顯著抑製.細胞遷移實驗結果 顯示,缺氧條件下榦擾Netrin-1的錶達顯著抑製HepG細胞遷移能力(P＜0.05).轉染 pGNET1後,HepG2細胞骨架髮生重組.結論 缺氧促進HepG2細胞骨架重組與Netrin-1錶達水平升高相關,Netrin-1錶達的上調可能是缺氧促進肝癌細胞骨架重組進而促進侵襲轉移的機製之一.
목적 연구결양대인간암세포HepG2세포골가중조적영향급Netrin-1재세포골가중조중적작용.방법 통과1%O_2 저양배양건립인간암세포HepG2물리결양모형,관측결양대세포형태적개변.구건침대Netrin-1 mRNA적간우질립pshRNA-Netrin-1급음성대조질립pGensil-1,병장기전염HepG2세포,사선은정전염세포주.관측전염pshRNA-Netrin-1질립전후,귀필배태검측결양대간암세포대HepG2세포골가중조적영향,세포천이실험검측결양조건하간우Netrin-1대세포천이능력적영향.동시구건Netrin-1진핵표체질립pGNET1전염HepG2세포,관측Netrin-1대간암세포골가중조적작용.결과 저양배양HepG2세포포체변득협장,세포련접송산.Netrin-1단백표체수평현저승고(P＜0.05).HepG2세포은정전염pshRNA-Netrin-1후,Netrin-1 mRNA표체현저하강.억제솔체73.32%±4.12%(P＜0.01).귀필배태검측F-actin결과 현시,결양촉진HepG2세포골가중조,전염pshRNA-Netrin-1후,결양촉진HepG2세포골가중조수도현저억제.세포천이실험결과 현시,결양조건하간우Netrin-1적표체현저억제HepG세포천이능력(P＜0.05).전염 pGNET1후,HepG2세포골가발생중조.결론 결양촉진HepG2세포골가중조여Netrin-1표체수평승고상관,Netrin-1표체적상조가능시결양촉진간암세포골가중조진이촉진침습전이적궤제지일.
Objective To study the effect of Netrin-1 siRNA on cytoskeletal arrangement induced by hypoxia in HepG2 cells.Methods HepG2 cells were cultured in 21% O_2 and 1% O_2.Morphological changes were observed after hypoxia treatment.The siRNA expression vector pshRNA-Netrin-1 targeting the mRNA of Netrin-1 and vector pGensil-2(as a control)were constructed,and then transfected into HepG2 cells.The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-Netrin-1 induced by hypoxia was analyzed by phalloidin.The migratory ability of HepG2 cells transfected with pshRNA-Netrin-1 induced by hypoxia was analyzed by cell migration assay.The Netrin-1expression vector was constructed and transfected into HepG2 cells.The cytoskeletal arrangement of HepG2 cells was analyzed likewise.Results hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment.Netrin-I expression increased in hypoxic HepG2 cells.Netrin-1 mRNA level was decreased by 73.32% ±4.12%(P＜0.01)after the pshRNA-Netrin-1 transfection.Hypoxia induced cytoskeletal arrangement of HepG2 cells.However,cytoskeletal arrangement of HepG2 cells transfected with pshRNA-Netrin-1 induced by hypoxia was inhibited in 1% O_2.As cell migration assay showed,the migratling number of HepG cells transfected with pshRNA-Netrin-1was significantly lower than that of control(P＜0.05).Netrin-1 could promote cytoskcletal arrangement of HepG2 cells by transfeeting Netrin-1 exprassion vector.Conclusion The expression of Netrin-1 in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of hepatocellular carcinom cells induced by hypoxia.Up-regulation of Netrin-1 expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.
