Study On Mechanisms And Signal Channels Of The Re-expression Of The Human Blood Coagulation Factor Ⅷ In Vivo Activated By The L-arginine In Vitro

Objective Adding L-arginine to the culture medium of human livercells L02in vitro, to make L02re-express endogenous coagulation factorVIII (coagulation factor VIII, FVIII), and then Study on mechanisms andsignal channels of the re-expression of the human blood coagulation factorVIII in vivo activated by the L-arginine in vitro.Methods the cells L02were divided into experimental group and blankcontrol group, the inhibitor group and the control inhibitor group; theexperimental group was added with a final concentration of15mmol/LL-arginine RPMI1640medium2ml, blank control group was added anequal volume of medium containing sterile water, the inhibitor group wasadded with afinal concentration of100mM nitric oxide synthase inhibitorL-NAME before adding L-arginine3hours, the control inhibitor group wasadded with a final concentration of100mM L-NAME RPMI1640medium2ml, respectively, culture0h,12h,24h,36h,48h,60h. Transcript levels ofhuman FVIII gene、iNOS gene、HNF1A gene、HNF4A genes、C/EBP alphagene、NF-kappaB gene、I-kappaB alpha gene、CREB gene and sGC alpha3 gene were detected by RT-PCR method; use Western Blot to detect theexpression of human FVIII、conventional I-kappaB and phosphorylation ofI-kappaB； PepTag Assay for Non-Radioactive Detection ofcAMP-Dependent Protein Kinase detect the change of level of PKAphosphorylation in experimental system; Dual Luciferase Reporter AssaySystem detect the transcriptional activity of FVIII promoter upstreamregulatory sequences; MSP (methylation special pcr) assay methylation’slevel of FVIII promoter upstream regulatory sequence.Results RT-PCR show that the experimental group has thetranscription of human FVIII mRNA in L02cells, blank control group、theinhibitor group and the control inhibitor group have no transcription ofhuman FVIII mRNA, the transcript levels of iNOS、HNF4A、NF-kappaB、I-kappaB alpha and CREB gene are rising and the transcription levels ofHNF1A, sGC alpha3and C/EBP alpha are decreased in the experimentalgroup, the transcription levels of these genes in blank control group、theinhibitor group and the control inhibitor group do not change significantly;Western blot show that after adding L-arginine, human FVIII express andexpression of phosphorylation I-kappaB is significantly increased, othergroups have no such change; PepTag Assay for Non-RadioactiveDetection of cAMP-Dependent Protein Kinase detect the levels of PKAphosphorylation in experimental system are increased; Dual LuciferaseReporter Assay System detect the transcriptional activity of FVIII promoter upstream regulatory sequences is increased; MSP (methylation special pcr)assay methylation levels of FVIII promoter upstream regulatory sequenceare decreased, demethylation levels are significantly increased.Conclusion On the one hand L-arginine activates MAPK signalingpathways by NO-cGMP-PKG signaling pathway leading to the changes inthe expression of human FVIII gene promoter upstream regulatory-relatedtranscription factors to activate the expression of human FVIII in humanliver cells L02; on the other hand, L-arginine activates the expression ofhuman FVIII by NO stress-induced demethylation of human FVIII genepromoter upstream regulatory sequences.