NO信号通过I-kappaB磷酸化激活人肝细胞内源凝血因子Ⅷ重表达的调控作用

Regulatory effect of NO signaling on expression of human endogenous coagulation factor Ⅷ by phosphorylation of I-kappaB

目的:建立一氧化氮(NO)前体化合物L-精氨酸激活人肝细胞L02内源凝血因子Ⅷ(FⅧ)重表达的分子细胞生物学模型,探讨NO信号激活L02中内源FⅧ重表达的调控通路及分子基础。方法:取对数生长期L02细胞随机分为对照组、加药组(L-精氨酸)、抑制剂组(L-NAME和L-精氨酸)和抑制剂对照组(L-NAME),分别培养12、24、36、48和60h,采用流式细胞术检测作用48h后L02中人FⅧ蛋白的表达,Griess法检测不同时间点L02细胞内NO水平,RT-PCR法检测L02中人FⅧ基因、诱导型一氧化氮合酶(iNOS)基因、核转录因子(NF-κB1)基因和核因子κB(免疫球蛋白κ轻链基因增强子)抑制蛋白α亚基(I-κB alpha)基因的转录水平,Western blotting法检测L02中人磷酸化I-kappaB(磷酸化I-κB)表达水平。结果:流式细胞术检测,加L-精氨酸培养48h后L02中出现人FⅧ蛋白的表达。Griess法检测,加药组L02在3、6和12h内NO表达水平显著增加(P<0.05),随后又基本恢复到正常水平,正常组、抑制剂组和抑制剂对照组L02细胞内NO水平无变化;RT-PCR检测,加药组L02中人FⅧmRNA转录;对照组、抑制剂组和抑制剂对照组均无人FⅧmRNA的转录,加药组iNOS、NF-κB1和I-κB alpha转录水平均上升(P<0.05),对照组、抑制剂组和抑制剂对照组上述基因转录水平均无明显变化。Western blotting法检测,加入L-精氨酸后,磷酸化I-κB表达水平明显增加(P<0.05),其他组则无此变化。结论:L-精氨酸通过NO信号通路进而激活I-κB磷酸化,导致人FⅧ基因启动子上游调控相关的转录因子NF-κB1进入胞核从而激活人离体肝细胞L02中内源人FⅧ的表达。

Objective To set up the molecular cytobiological model of endogenous coagulation factor Ⅷ （FⅧ） re-expressing in human liver cells L02,and to study the regulation pathway and molecular basis of the re-expression of FⅧ in L02 cells activated by NO signal.Methods The L02 cells at logarithm growth phase were selected and＆amp;nbsp;randomly divided into blank control group and experimental group, inhibitor group and inhibitor control group;they were cultured for 0,12,24,36,48,and 60 h.Flow cytometry was used to detect the expression of human FⅧ protein in L02 cells after treated for 48 h.Griess experiment was performed to detect the levels of NO in L02 cells at different time points;the transcription levels of human FⅧ gene,iNOS gene,NF-κB1 gene and I-κB alpha gene were detected by RT-PCR method.Western blotting method was used to detect the expression levels of human phosphorylated I-kappaB （phosphorylated I-κB）in L02 cells.Results The results of flow cytometry showed that the expression of human L02 FⅧ protein was found after treated with L-arginine for 48 h. The Griess results showed that the levels of NO in L02 cells in experimental group were significantly increased at 3,6,12,and 24 h （P〈0.05）and the levels of NO in blank control group,inhibitor group and inhibitor control group had no changes. The RT-PCR results showed that the transcription of human FⅧ mRNA in L02 cells was found in experimental group,but there was no transcription of human FⅧ mRNA in blank control group,inhibitor group and inhibitor control group;the transcription levels of iNOS,NF-κB1 and I-κB alphain experiment group were increased（P〈0.05）and the transcription levels of these genes in blank control group,inhibitor group and inhibitor control group had no changes. The Western blotting results showed that after adding L-arginine the expression level of phosphorylated I-κB was significantly increased （P 〈 0.05 ）, other groups had no such change. Conclusion L-arginine can activate the phosphorylation of I-κB by NO signal pathway to lead to the changes in the expression of human FⅧ gene promoter upstream regulatory-related transcription factors NF-κB to activate the expression of human FⅧ in human liver cells L02.