放线菌素D/TNF-α诱导大鼠肝干细胞凋亡及HGF的拮抗作用

Liver stem cell apoptosis induced by ActD/TNF-α and inhibited by HGF

目的 研究TNF α、肝细胞生长因子 (HGF)在肝干细胞的增殖、分化调控及凋亡中的作用。方法 用大鼠肝干细胞系WBF 344细胞进行实验 ,用MTT法检测细胞毒作用 ;用DNA凝胶电泳及流式细胞仪检测细胞凋亡 ;用Westernblot方法检测磷酸化MAPK及PI3K蛋白的表达。结果 发现TNF α单独对WBF 344细胞无明显作用 ;而TNF α对放线菌素D(ActD)致敏的WBF 344细胞有明显细胞毒作用。进一步 ,发现TNF α能够诱导ActD致敏的WBF 344细胞发生凋亡 ,在作用 9h即出现细胞凋亡 (13.6 0 % ) ,4 8h达高峰 (5 1% ) ,并且这种凋亡呈剂量效应关系。我们还发现 ,HGF可以明显拮抗ActD/TNF α诱导的WBF 344细胞凋亡 ,并且呈剂量效应关系。Westernblot结果显示 ,HGF刺激后 ,磷酸化MAPK蛋白高表达 ,表明HGF激活了MAPK途径。我们进一步用PI3K特异的抑制剂WT阻断PI3K途径 ,发现HGF的抗凋亡作用被阻断 ;然而 ,用MAPK特异抑制剂U0 12 6阻断MAPK途径后 ,却并不影响HGF的抗凋亡作用。结论 TNF α能诱导ActD致敏的肝干细胞发生凋亡 ,而HGF则对这种细胞凋亡有明显的拮抗作用 ;虽然HGF能够激活PI3K及MAPK 2条途径 ,但其抗凋亡作用是由PI3K途径传导的。

Objective To investigate the anti apoptosis effect of HGF on ActD/TNF α induced apoptosis in a liver stem like cell line WB F 344. Methods WB cell were exposed to ActD, TNF α or/and HGF. The cytotoxic effects were tested by MTT; apoptosis were examined by flow cytometry (FCM) and DNA fragmentation; the phospho MAPK were detected by Western blot. Results The results showed that ActD/TNF α could induced apoptosis in WB F 344 cells, but TNF α alone couldn′t induced a significant apoptosis. The apoptosis was found at 9?h after adding ActD/TNF α and approached to the highest at 48?h. Furthermore, we found that HGF significantly reduce the apoptosis of WB cell induced by ActD/TNF α and enhanced with increase of HGF concentration. Our results also showed that the anti apoptosis effects of HGF were inhibited when PI3k pathway was blocked by the specific inhibitor Wartmanner, but not inhibited by blocking MAPK pathway. Conclusion Apoptosis of WB cells can be induced by ActD/TNF α, and HGF can protect the WB cells from ActD/TNF α induced apoptosis. Although HGF can activate both PI3K and MAPK pathways, the anti apoptosis effect of HGF occurs via a PI3K pathway and is not dependent on the MAPK pathway. [