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目的初步探讨神经生长因子Netrin-1对人肝癌细胞HepG2抗失巢凋亡的作用及相关机制。方法采用脂质体转染的方法,将Netrin-1的真核表达质粒转染到肝癌细胞HepG2中,Western blot检测Netrin-1的表达;用软琼脂集落试验、悬浮培养模型及流式细胞检测的方法观察肝癌细胞HepG2抗失巢凋亡的特性。结果肝癌细胞HepG2在失巢状态下可以聚集成团生长,并与贴壁生长有相似的凋亡比例。与对照组细胞相比,稳定转染Netrin-1真核质粒的HepG2细胞在软琼脂中形成细胞集落的数量明显增加,在悬浮培养模型中形成细胞团的直径更大,在失粘附状态下的细胞凋亡比例明显降低;磷脂酰肌醇3-激酶(PI3K)通路的抑制剂LY294002能增加转染细胞在失粘附状态下的凋亡比例。结论肝癌细胞HepG2具有抗失巢凋亡的特性,Netrin-1可以增强HepG2细胞抗失巢凋亡的能力,其机制可能与PI3-K通路相关。
Objective To investigate the effect of Netrin-1 on anoikis and its related mechanisms in HepG2 cells. Methods The eukaryotic expression plasmid of Netrin-1 was transfected into HepG2 hepatoma cells by lipofection method. The expression of Netrin-1 was detected by Western blot. The expression of Netrin-1 was detected by soft agar assay, suspension culture and flow cytometry Methods To observe the anti-anoikis characteristics of HepG2 cells. Results HepG2 cells could aggregate into clusters in the state of anoikis and had a similar percentage of apoptotic cells. Compared with control cells, HepG2 cells stably transfected with Netrin-1 eukaryotic plasmid showed a significant increase in the number of colonies formed in soft agar, larger diameters of cell clusters formed in the suspension culture model, and in the absence of adhesion state (P <0.01). The ratio of apoptosis in transfected cells was significantly decreased by LY294002, a PI3K pathway inhibitor. Conclusion HepG2 cells have the anti-anoikis function. Netrin-1 can enhance the anti-anoikis ability of HepG2 cells, which may be related to PI3-K pathway.