论文部分内容阅读
目的:在建立高纯度小鼠肝血窦内皮细胞的体外培养的基础上研究γ分泌酶抑制剂(DAPT)对肝血窦内皮细胞活性的影响。方法:首先通过胶原酶灌注消化、percoll梯度离心和选择性贴壁分离得到高纯度、可在体外条件下培养的肝血窦内皮细胞,其次用不同浓度的DAPT(15μmol/L、45μmol/L、75μmol/L)处理细胞,然后通过MTT检测细胞增殖情况、Real time PCR检测相关分子改变。结果:在体外条件下DAPT对肝血窦内皮细胞的增殖起到促进作用,这种促进作用随着DAPT浓度的增加相应的增加;DAPT能够导致肝血窦内皮细胞Notch信号下游分子Hes1表达下调,VEGF信号中VEGFR1表达下调,VEGFR2表达上调。结论:γ分泌酶抑制剂(DAPT)通过抑制肝血窦内皮细胞Notch信号,引起肝血窦内皮细胞表面VEGFR1表达下调,VEGFR2表达上调显著增加肝血窦内皮细胞的活性。
OBJECTIVE: To study the effect of γ-secretase inhibitor (DAPT) on the activity of hepatic sinusoidal endothelial cells in vitro on the basis of establishing a high purity mouse hepatic sinusoidal endothelial cell. Methods: Liver sinusoidal endothelial cells with high purity and cultured in vitro were obtained by collagenase perfusion digestion, percoll gradient centrifugation and selective attachment separation, followed by different concentrations of DAPT (15μmol / L, 45μmol / L, 75μmol / L). Then the cell proliferation was detected by MTT and the related molecular changes were detected by Real time PCR. Results: In vitro, DAPT could promote the proliferation of hepatic sinusoidal endothelial cells, and the promoting effect increased with the increase of DAPT concentration. DAPT could down-regulate the expression of Hes1 in Notch signaling of hepatic sinusoidal endothelial cells, VEGFR1 expression in VEGF signaling was down-regulated and VEGFR2 expression was up-regulated. CONCLUSION: γ secretase inhibitor (DAPT) can downregulate the expression of VEGFR1 on the surface of hepatic sinusoidal endothelial cells by inhibiting the Notch signal of hepatic sinusoidal endothelial cells. The upregulation of VEGFR2 expression significantly increases the activity of hepatic sinusoidal endothelial cells.