胆道探查致十二指肠对穿伤、腹膜后结石遗留一例

Effect of Netrin-1 on cytoskeletal arrangement induced by hypoxia in HepG2 cells

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.

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% O2 and 1% O2.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.